AU616696B2 - Adrenal response in animals - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class 1I. C 9 Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art:

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APPLICANT'S REFERENCE: CAP of PI 5140 *4 i Ir Name(s) of Applicant(s): Ciba-Geigy Australia Ltd Address(es) of Applicant(s): 140 Bungaree Road Pendle Hill New South Wales Australia Address for Service is: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Complete Specification Cor the invention entitled: ADRENAL RESPONSE IN ANIMALS Our Ref: 73270 POF Code: 1422/50647 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 1 _III 1_ 2 The present invention relates to a method for the testing of reaction of animals to stress-related stimuli and to the selection of animals for breeding on the basis of test results.

The need for objective methods of evaluating an animal's response to stressors is well recognized. An animal's response to stressors has important implications for animals subjected to intensive husbandry conditions.

Commercial farm animals are constantly exposed to a wide range of external or stressful stimuli. In all animals one of the first defences against these stiiuili is a heightened activity of hypothalamic-pituitary-adrenal axis.

This heightened activity leads to an increase in the secretion of hormones, called glucocorticoids, from the adrenal gland. Such e response is essential if the animal is to be able to adapt to that stimuli and survive without detriment to its well being. However it is well documented that an excessive or prolonged response to persisent or recurring stimuli can result in a reduction in that individuals well-being. For example reproductive capacity may be impaired, growth rate of growing animals reduced or the animals immunity to diseases reduced.

Various techniques of evaluation have been employed.

For instance, in the pig, plasma concentrations of various hormones and enzymes have been measured separately or in combination in an attempt to assess objectively the animals' response to stress. It has been suggested that catecholamine levels could be used to measure the response o. to the stress of sampling, corticosteroid levels to indicate the degre of adaption to long term stressors and to evaluate the extent of acute stress response sampling trauma), and enzyme levels to measure the reaction to stressors experienced in the previous 24 hours. The most oo commonly used system for the evaluation of stress responses is the plasma levels of hormones secreted by pituitary and adrenal gland. However, diurnal variations and episodic fluctuations in hormone levels should be taken into consideration. Annual changes in sensitivity of the adrenal 3 glands, and the variations introduced by the stress of handling, further complicate the interpretation of the results. The necessary precautions to avoid the influence of factors such as diurnal and annual variations, the stress of handling, the blood sampling make this type of stress evaluation unsuitable for commercial herds.

Another possible approach to the evaluation of an individual animal's "excitability" or "sensitivity" to stressors is to evaluate the individual's response to S 10 external stimuli or stressors. Perception of and response to a stressor will be reflected by increased levels of ACTH and in turn elevated levels of corticosteroids. However, this reaction is non-specific and its magnitude depends on the sensitivity of the hypothalamus, pituitary and adrenal glands.

It is accordingly an object of the present invention to overcome, or at least alleviate, one or more of the difficulties and/or deficiencies related to the prior art.

Accordingly, in a first aspect, there is provided a method for testing the reaction of animals to stress-related o stimuli which method includes 0 providing an animal to be tested, and a predetermined oo. amount of an adrenal stimulant; administering the adrenal stimulant to the animal; and measuring the adrenal response after a S predeterminod interval of time.

It hai been discovered in accordance with the present o invention, that when an adrenal stimulant is introduced to a Sgroup of similar animals, due to their inherent variation in 3 their adrenal glands, some animals will mount a large 0 a 0 response and secrete large amounts of glucocorticoid hormones, while others will have a lesser response and S secrete low amounts of glucocorticoids.

o o The measurement of adrenal response may comprise a o measurement of the corticosteroid level in the blood of the S animals. By taking a blood sample at a predetermined time after the injection, it is possible to measure the glucocortiloid hormone concentration and thus classify r -4individual animals as potential high, medium or low responders to stressful stimuli.

The animals which may be subject to the stress test may be of any suitable type. Commercial farm animals for example may be used. For example, bovine, porcine, ovine and avian animals may be subjected to the stress test.

Particular reference is made to' porcine animals below but this should be understood to be illustrative only.

The adrenal stimulant utilised in the method for testing of stress related reactions, may be of any suitable type. The adrenal stimulant may be a sugar or a fatty acid. The preferred adrenal stimulant is a hormone, An adreno-corticotrophic hormone (ACTH) may be used. This hormone is produced by the pituitary gland in response to stressful stimuli and it controls the release of glucocorticoids from the adrenal gland. The hormone consists of 39 amino acids, the first 24 of which are identical for all species of animal so far tested. It is these first 24 amino acids that are necessary for the biological activity of the hormone. Thus whilst a natural ACTH hormone may be used, a synthetically produced hormone comprising the first 24 amino acids will be biologically S..o active in substantially all species. The synthetic ACTH, Tetracosactrin, may be used.

.o A synthetic ACTH sold under the trade designation SYNACTHEN and available from Ciba-Geigy Australia Limited has been found to be suitable.

o The adrenal stimulant may be administered in any S" suitable manner. The adrenal stimulant may be introduced by *3 0" injection. An intravenous, subcutaneous, intramuscular, oral or similar administration may be used. The amount of adrenal stimulant given to the animal may vary with the 5 animal species. For a porcine animal, an amount of S. approximately 25 i.u. to 75 i.u. ACTH may be used.

0 t Preferably an intramuscular injection of approximately i.u. may be used. Such preferred dose allows for the possibility that in a field testing situation where all animals may not be at "resting basal" conditions when injected, as they were in these experiments, it was necessary to provide maximal stimulation of the adrenal gland to be sure that the differences seen in adrenal response were an accurate reflection of that animal's potential.

The measurement of adrenal response may be taken at any suitable but standardised interval of time. Peak response for porcine animals appears to occur in the range of approximately 50 to 70 minutes after initial injection.

Preferably, the predetermined interval of time for porcine animals is 60 minutes after initial injection.

The step of measurement of corticosteroid levels in the blood sample may be undertaken in any suitable manner.

Any standard blood assay method may be utilised. An ELISA method or radioimmunoassay method may be used. For radioimmunoassay, a radioactive cortisol may be used. An iodine-labelled cortisol may be used. 12 5 I-labelled cortisol may be used. A double antibody radioimmunoassay is particularly preferred.

The double antibody radioimmunoassay may be conducted as follows: Plasma aliquots are incubated with a first and second antibody mixture, iodine-labelled cortisol is added and incubation continued at elevated temperature. A gamma globulin is added, the supernatant decanted and thprecipitate counted.

Antibodies may be generated in any suitable animal.

For example a rabbit may be used for immunisatlon with a suitable conjugate. To ensure an adequate response, a cortisol:protein conjugate may be used. A cortisol 3-methyl 03V oxime:bovine albumin conjugate may be used.

In a still further aspect of the present invention, the method of testing the reaction of animals may be adapted o 0 for on-site evaluation, In this aspect the step of o a measurement of corticosteroid levels in the blood sample after a predetermined interval of time may utilise an o enzyme-linked immunosorbent assay method (ELISA method).

An ELISA method has the advantage that a blood sample may be tested directly and simply utilising an ELISA test 6kit as described below.

In accordance with this aspect of the present invention there is provided a diagnostic test kit including a vessel suitable for use in an enzyme-linked immunosorbent assay, an antibody against cortisol in a suitable container, an enzyme-linked antibody conjugate in a suitable container, an enzyme substrate in a suitable container.

The vessel may be a microtitre plate. The microtitre plate may be manufactured in polystyrene or similar material.

The antibody may be generated in any suitable animal.

A rabbit may be used. To ensure an adequate response, a cortisol:protein conjugate may be used to immunise the animal. A cortisol 3-methyl oxime:bovine allumine conjugate may be used.

In a preferred form, the anti-cortisol antibody may be coated on a portion of the vessel. Where the vessel is a microtitre plate, the anti-cortisol antibody may be coated on a plurality of the walls thereof.

r The enzyme may be selected from any of those suitable °oo, for use in an ELISA test method. The enzyme may be selected from alkaline phosphates, glucose oxidase, a horseradish peroxidase or urease.

*0 The selection of enzyme substrate will be related to S. the selection of enzyme as is known per se. For example for the enzyme horseradish peroxidase, an ABTS or an O-toludine olution may be used. For urease a standard urease S substrate solution may be used.

The results of the ELISA test may be measured i utilising any suitable visual or mechanical means as known S per se. These may be compared to a standard if required.

Examples of two different enzyme labels are the conventionally used peroxidase and urease. Urease activity can be detected using a pH indicator (bromocresol purple) and gives a change from one intense colour (yellow) to another (purple), rather than the gradual increase in colour obtained when using an ehzyme such as peroxidase. This 7 gives sharper, more clearly, visually distinguishable reaction end-points.

Further any suitable assay style may be used in the ELISA method. Examples of two styles are as follows: 1) Enzyme-labelled Cortisol Anti-cortisol IgG is immobilised on the wells of microtitre plates. Cortisol is estimated by the competition between cortisol in serum samples and enzyme-labelled cortisol for the immobilised IgG.

Cortisol-peroxidase conjugate. This may be prepared using a mixed anhydride reaction and the cortisol-3-CMO derivative. The most effective well coating is obtained using a glutaraldehyde pretreatment of the IgG. Assay conditions which may be used are a 1 hour incubation at room temperature followed by an enzyme reaction time of minutes. Three preferred peroxidase substrates include OPD, ABTS, TMB. When using a microtitre plate reader, pig serum samples can be easily distinguished into high, medium and low responder groups with good correlation between results from the ELISA and the "in-house" RIA currently being used.

2) Enzyme-labelled Antibody This procedure uses protein-conjugated cortisol immobilised on a microtitre plate. It depends on cortisol 0 0 present in serum samples decreasing the binding of o o o anti-cortisol-IgG to the immobilised cortisol. The anti-cortisol IgG can be enzyme-labelled or its binding can be detected using labelled anti-rabbit IgG.

So oo The assay was set up using commercially available S urease anti-rabbit IgG. With both NUNC Immunoplate I and COSTAR Fastbinder plates cortisol-BSA was more effective o than cortisol-ovalbumin for well-coating. The assay S conditions were optimised within the constraints of 1 hour 4 incubations at room temperature. The cortisol dose-response o 0: curve obtained may be used for visual discrimination of 0 serum samples into low, medium and high responder groups.

a0soe4 It will be recognised, that once animals are given an adrenal stimulant, and then glucocorticoid response measured, as described above, they may then be classified as r 8 potential high, medium or low responders to stressful stimuli. It is then possible to produce a line of animals with a low potential response to stressful stimuli. Such animals are then genetically and physiologically more suitable for intensive husbandry conditions.

Accordingly, in a further aspect, the present invention provides a method for reducing the adverse response to stress-related stimuli of animals, which method includes providing a plurality of animals; subjecting the animals to the method of stress-testing described above and selecting animals exhibiting a low adrenal response.

The plurality of animals to be tested may be of a similar age, sex and body weight. The method for reducing the adverse response of animals according to this aspect of the present invention may further include the further step of: subjecting the selected animals to a selective breeding program.

It will be understood that the selective breeding step Smay be repeated, if desirel, to increase the proportion of each animal flock exhibiting a low response to stressful 0 UJ stimuli. Such breeding program provides a high percentage o 0 of offspring exhibiting a similar low adrenal response rate. Ultimately this will be reflected in improved °9 performance of the husbandry enterprise.

Accordingly, in a still further aspect, the present invention provides a line of animals exhibiting an inherently low response to stress related stimuli. As described below, such a line of animals has been shown to exhibit a heritable low adrenal response rate in a high percentage of cases.

o 09 The line of animals exhibiting heritable low adrenal response to stress related stimuli may be a line of porcine a animals.

0*40 The data presented here show that a population of pigs vary in their adrenal response to challenge with a standard dose of AC.'H and that the type of adrenal response is 9 consistent and repeatable within animals, i.e. pigs may have an inherent high, low or average adrenal responsivenes to challenge with ACTH. Based on this we have developed a dynamic test to detect the high and low responding extremes in the population, a test that is repeatable and is not affected by short term exposure to ACTH. Furthermore, the individual pigs adrenal response to ACTH has been shown to be an accurate reflection of that individuals adrenal response to stress. We have also clearly demonstrated that there are considerable economic benefits to be gained by selecting for pigs that have a low adrenal responsiveness.

The present invention will now be more fully described with reference to the accompanying experiments. It should be understood, however, that the discussion following is illustrative only and should not be taken in any way as a restriction on the generality of the invention as decribed above.

MATERIALS AND METHODS Cortisol Radioimmunoassay oa o Plasma cortisol concentration was measured using a S double antibody radioimmunoassay. Replicate analysis of two pools of normal pig plasma (139.9 18.1 nmol/l n=42, mean sd and 71.2 7.7, n=50) showed an inter-assay co-efficient o of variation of 12.9 and 10.8% respectively. The 0 intra-assay variation for 12 replicates of these two pools was 3.2 and 2.7% respectively. Twenty duplicates of the zero standard (maximum binding) were assayed in a single S run. The sensitivity of the assay was set at 2 standard ,~aO c deviations below the mean of these replicates, and found to be 5 nmol/l.

*o*o To examine the accuracy of the assay plasma samples with various levels of cortisol were assayed using three different systems. Aliquots of 21 samples were assayed by 0 "o double isotope dilution derivative assay (DIDDA) and in our RIA system. The correlation coefficient between the two sets of assays was r 0.97; and the equation to the regression line was y (DIDDA) 0.77x 7.45. Another set 10 of 15 samples was assayed simultaneously using our RIA system and a commercial cortisol kit (Cortisol Autopak, Micromedics, The kit was a direct, solid-phase method 125 with I-labelled cortisol. The correlation coefficient for the two months was r 0.94 and the equation to the regression line was y (Micromedics) 0.97x 0.10.

Experiment 1: Variation in Adrenal Responsiveness Landrace and Large White/Landrace cross female pigs aged 14 weeks were used for this experiment which was conducted on a commercial piggery. The Landrace group consisted of 15 treated animals and 8 controls and the Large White/Landrace cross had 15 treated and 10 control animals.

Animals were randomly allocated to treatment or control groups from a large pool of gilts. Each pig was identified, bled by jugular venepuncture, and injected intramuscularly with either 25 i.u. Tetracosactrin, (Synacthen, Ciba-Geigy Australia Ltd.) treatment group or physiological saline control group. One hour later all animals were re-bled.

Plasma was separated by centrifugation and kept frozen (-200C) until assayed.

°o Resting concentrations of cortisol were not different between the breeds (P Similarly, mean cortisol 0 0 o levels were not different before and after saline injections ooo in both breeds (P Cortisol levels after ACTH challenge were different from resting cortisol levels I (P .01) in both breeds of animals, and there was no difference in the magnitude of the response between the breeds (P .05) (Table The adrenal response of S individual animals to ACTH challenge revealed up to a 0"o' fivefold difference in post-challenge cortisol levels, Experiments 2 ayld 3: 000040 S a) Animals oer04e Two groups each of 15 female pigs of Large White X Landrace origin aged about 12 weeks and of similar live ,to a weights (approx. 45kg) were used in these experiments.

These pigs were selected from two groups, each of 40 pigs on a commercial herd on the following basis. Each pig was identified, blood sampled by jugular venepuncture, and l-----rpurrr-- 1~ 11 injected with 25 i.u. synthetic ACTH. One hour later each pig was re-sampld and the cortisol concentration of the plasma determined by radioimmunoassay. Each group of experimental animals was selected to ensure that the subgroup of 15 was representative of the spread of adrenal responsiveness in its population of 40. The selected animals were transported to the laboratory where they were housed in individual wire mesh stalls. They were fed approximately 2.0kg of commercial grower pellets daily and had ad libitum access to water. A 3 week familiarization period was allowed before to the commencement of experiments.

b) Cannulation The pigs used in experiments 2 and 3 were then sedated with Stresnil (Ethnor, North Ryde, 1 ml per 20 kg body weight). Indwelling polyvinylchloride cannulae (1.5 mm OD; 1.0 mm ID; 0.5 m length) were inserted into a marginal ear veir via a 50 mm 14 g needle. The needle was removed and the cannula sutured in place. A small cloth purse was glued and sutured to the pig's ear to house the cannula when not in use. The cannula were flushed daily with heparinized a saline (100 i.u./ml).

c) Experimental Designs S Exppient 2: Optimum Dose and Sampling Time The first of the two groups of 15 animals (pigs s numbered 1-15) was used in this experiment which was a "oS designed to establish the most practical dose of ACTH and the best time to sample after challenge such that the o0, extreme responders in the population could be reliably detected. The doses of ACTH used were 2.5, 5, 10, 25 and i.u. given as a single intramuscular injection. Each animal received the same dose of ACTH on the same day, with a 3 day rest between injections. Blood samples were taken from all animals 60 and 0 min. prior to the ACTH injection (at 0900 S h) and 15, 30, 45, 60, 75 and 90 min., 2, 3 and 4 h after injection of ACTH.

S Experiment 3: Repeatability of Adrenal ResponsiVenes The following experiments were done on the second group of 15 pigs (numbered 16-30) to examine the -12repeatability of adrenal responsiveness.

Each animal was challenged with ACTH on up to five occasions over a 3 week period, using the dose established in experiment 1, with 3 days rest was between challenges. Blood samples were taken 60 min.

before, and 0, 45, 60 and 75 min. after ACTH challenge.

Pigs were bled 60 min. before challenge with the dose of ACTH established in experiment 1 and again 0, 1, 2, 3, 4 and 5 hr after challenge. Immediately following the last (5 hr) blood sample the pigs were rechallenged with another dose of ACTH. They were bled 45, 60, 75, 90 and 120 minutes after the second challenge.

Pigs were bled 60 min. before challenge with ACTH and 0, 45, 60, 75, 90, 105, 120 min. 2.25, 2.5, 2.75, 3, 3,25, 4, 5, 6, 6.5, 6.75, 7, 7.25, 7.5 and 8 h after the initial challenge. Additional challenges of ACTH were given 1, 2 and 6 h after the initial challenge.

RESULTS

*O*o For each of the two groups of animals, used in oo experiments 2 and 3 the 15 selected were representative of o the population from which they were chosen, In each case 04,006 the mean cortisol concentration 60 minutes after the 4 0 0 °i o o initial on-farm challenge with ACTH was not significantly j o different from the population from which they were selected i (307 97 and 279 77 P 0.147 for group 1 selecta and unselected animals respectively and 284 83 and 278 64 P 0.22 for group 2 selected and unselected 00 i) animals respectively.

S Experiment 2: Optimum Dose and Sampling Time The mean response of all animals to each of the five S doses of ACT;I is shown in Figure 1. For each animal and for the mean response of all animals the 2.5 i.u. dose resulted in sub-maximal stimulation, the response being lower and shorter than that with 5.0 i.u. which was lower and shorter than the 10.0 i.u. The 25 and 50 i.u. doses gave equivalent response curves and both appear to provide maximal -13stimulation. Over the range 2.5 to 25 i.u. the cortisol concentration at 60 min. was linearly related to the log of dose given (r 0.995). An example of the variation seen in adrenal response to ACTH between individual pigs can be seen in Fig. 2.

To simplify comparison of the individual responses between animals and across tests the data have been analysed on ranks from lowest to highest cortisol concentration. The data were analysed as the absolute peak concentration, the concentration at a particular time, as the area under the curve for the 3 h following challenge and as the peak increment above baseline. The ranking system (using each of these four methods) indicated that two animals were consistently ranked in the two lowest responders while another two animals were consistently in the highest. These four animals were also the extreme reactors from the first group of 40 pigs in the initial "on farm" selection test.

The optimum time to sample blood in order to detect the peak cortisol concentration in response to challenge varied with the dose. The two high and two low responding animals could just be detected at 30 minutes only with the and 5.0 i.u. doses, and at 45 minutes with the 10 i.u.

does. The extreme responders could readily be distinguished at 60 minutes with the higher two doses. They could also be readily detected at 45 and 75 min. with, i 50 i.u. dose.

SFrom a practical view point, it was decidw to base future tests on 50i.u. ACTH with the post-challenge blood samplas taken at 60 minutes. This dose was chosei because in providing maximal stimulation of the adrenal gland it reflected the potential for response of that individual.

iAlso it was envisaged that in figure experiments large numbers of animals may be challenged on farms. The o, dose provided some safety in the time at which the post challenge blood sample could be taken.

S Experiment 3: Repeatability of drenal Responsivaness o«o* Because of problems with the patency of cannulae not all pigs were available for all tests. Up to five identical tests were used over a 3 week period to examine the .c

I*

14 repeatability of adrenal responsiveness of individual pigs.

A moment co-efficient of skewness and kurtosis test showed that for each of the 5 tests the cortisol concentration of the individual pigs at 60 min. post challenge were normally distributed as was the population of 40 pigs from which these were selected. While the magnitude of adrenal response varied daily, the relative response between animals showed considerable uniformity over the five tests. The mean and standard deviation of each animals response to the five tests is shown in Table 2. The variance of the adrenal response was not homogenous between animals, and so the data were analyzed using non-parametric statistical tests. The cortisol concentration at 60 min. was ranked from lowest to highest for each test. On 5 out of 5 occasions pig number was ranked in the 2 lowest, while on 3 out of 3 and 4 out of 4 occasions pig numbers 20 and 21 respectvely wre ranked in the highest. Statistical analysis revealed highly significant differences in adrenal response between animals using a Kruskal-Wallis analysis of variance (P .001) and a high level of agreement in the rankings of the individual animals over the 5 tests using a Kendall's Co-efficient of 0,40 Concordance test .005). In the initial test of 40 pigs S from which these 15 experimental pigs were jelected the t hird highest responder was number 20, the highst responder a S was number 21 and the lower number In the first experiment to examine the short term repeatability of adrenal responsiveness the pigs were S challenged with 50 i.u. ACTH at 10am then again 5 h later.

S The mean response of all animals (n 5) and an example of the response of four individual animals is shown in Figure 3. In each animal the morning response was greater than the afternoon response. The average min. post challenge S cortisol concentration in response to the morning challenge waas 324 144 nmol/l/. The mean response to the afternoon S challenge was significantly lower (222 82 nmol/l) using a Student's t-test (P Figure 4 shows the time response curves following challenge with 50 i.u. ACTH at Oh, lh, 2h and 6 h. The mean a a I a

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~;..rlrr 15 cortisol concentration 60 min. after the first challenge was 281 95 nmol/l, this had increased significantly (P .001, using a Student;s t-test) to 380 150 nmol/l 60 min. after the second challenge. One h after the third challenge the co:tisol concentration was still 380 153 nmol/l/ When rechallenged 6 h after the initial dose, the average concentration after 60 min. was only 215 90 nmol/1 significantly lower (P<.001) than the level 60 minutes after second and third challenges and lower than the initial peak. More importantly the animals that gave the highest and lowest responses 60 min. after initial challenge gave, the highest and lowest responses to each of the subsequent 3 challenges. At no time did the cortisol concentration of the animals classified as low responders reach the concentration seen 1 hour after the initial challenge in the high responders nor did it reach the mean concentration shown by all pigs at 60 minutes after the initial challenge.

Experiment 4: Long term repeatability of adrenal o260 responsiveness Q 0 o A sample population of 120 female pigs of Large White X Landrace origin aged about 3 weeks were identified, bled, injected with 2.5 i.u. ACTH and rebled one hour later.

Three groups of four pigs were selected at random from those individuals that fell into the 10% of that population with the highest, lowest and median adrenal response. At about 18 weeks of age the 12 selected pigs were rechallenged with S* 50 i.u. ACTH.

Table 3 shows the pre and post challenge cortisol concentration for 12 pigs tested at about 3 weeks and 18 weeks of age. The type of response to ACTH was highly consistent over the 2 tests. (Rank-order correlation co-efficient r 2 0.8012 P <0.001).

6 0 w Ir I7 16 TABLE 1 Plasma cortisol levels (nmol/l) in L and rvF and Landrace/Large White cross pigs before and after stimulation with 25 i.u. of ACTH-.

Values are means Number of animals shown in parenthesis. Values with different superscripts are significantly different at che 5% level.

Cortisol Concentration Breed Treatment Pre- Post- Post-challenge challenge challengie Rangie 30.7 15 1 a 162.0 69 1 b 44.243 Landrance Saline(8) 26.4 7. a 29.4 +12.6 a Large White ACTH(15) 2 9 .4 9 9 a 154.9 65 6 b 40.234 Landrace Cro~ss Saline(lO) 3 9 .6 13. 2 a 4 3 .2 +12. 9 a 0 00: 0 0 0 0069004 4 0 0 00 0 00 b0 00 0 0 0 2 0

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4 0 0 ~0 oeooe4 4 0 000000 0 4 0 00 06 0 *04 0 4000 j 0 00 17 TABLE 2 mean plasma cortisol concentration (nmol/l) 60 min.

after intramuscular challenge with 50 i.u. ACTH on up to separate occasions. (Mean standard deviation, number of tests in parentheses).

Animal Cortisol Range Number Concentration 16 357 177 284-569 17 183 51 132-227 18 169 ±19 149-186 19 283 ±~61 226-357 589 ±163 452-769 21 518 ±39 468-552 22 268 ±52 202-328 23 410 ±128 249-600 24 250 ±37 208-280 154 ±22 127-178 26 250 ±50 171-305 27 248 ±51 189-230 28 272 ±55 208-304 29 292 ±75 225-420 30 327 ±108 217-447 00 0 0 4 I 60 0 04 00 4 0 60 4 0~ 0006 0 0 0 o. 0300 0 0 0 00 00 0 300 0 0 00~00.

0 0 r i 18 TABLE 3 Plasma cortisol concentrations challenged with ACTH at 3 and 18 weeks (nmol/l) of age.

in 12 pigs Animal Ni imh nr 732 795 810 924 251 46 536 646 690 2 357 652 3 weeks old Pre ACTH Post ACTH- 56 510 102 562 78 516 63 725 92 337 117 315 62 343 48 319 ill 180 123 204 69 184 57 174 18 weeks old Pre ACTH Post ATCH 189 715 ill 553 90 495 214 726 68 530 392 271 389 207 395 229 268 88 239 130 182 76 136

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44 4 4 4 a a A 4044 044 Rank order correlation co-efficientr2 challenge cortisol concentrations.

0.8012 for post -19 Experiment 1. Heritability and Superior Growth/Feed Conversion For the growth/feed conversion study the pens avaiLlable held on average 22 animals. From the previous experiment is was decided that 4 replicates of each responder type was necessary. thus if we wished to take the top, median and bottom 15% of the population we needed to test approximately 1200 piglets. To achieve this number we had to test 6 piglets at 2-3 weeks of age from 100 litters on each of 2 consecutive weeks. The piglets were weighed when tested and those selected for the growth trial were reweighed and allocated randomly within sub-groups into pens of about 22-23. These pigs were weighed individually when moved to the grower shed at 9 weeks of age and again at to 21 weeks when moved into and out of the finisher shed.

The amount of feed allocated to each pen during the grower and finisher stages was monitored and pen feed conversion calculated.

A summary of the results of the growth/feed conversion .ZOstudy is shown in Table 4 and Figures 5 and 6.

099 The following points should be noted from Table 4.

There appears to be differences in growth rate and fesed conversion between weeks.

S The low responders were heavier at 2-3 weeks of age :00and grew at a f aster rate than the medium and high responders.

Differences between high and low responders seems 0 00 consistent between males and females.

o A preliminary 2 way ANOVA (using only groups and week,- in the analysis) showed significant differences 9@O~ between weeks for the females and highly significant differenices between the high, medium and low responder groups for both male and females inthe final pre-sale weight and in the total gain during the study (Table 4).

The feed conversion data is also summarized in Table Is4 4. There are significant differences in the Feed conversion between weeks (P <0.001) and between responder groups (P 0.01) for both males and females. It should be r I 1 emphasised that the feed conversion data are calculated for a fixed time period and thus we have probably over estimated the actual feed conversion rate of the medium and high responders. If all pigs had been "finished" to the same weight then the medium and high responders would have eaten substantially more feed for the same weight gain.

The heritability of adrenal responsivenss was estimated using the 1/2-sib analysis method. Approximately boars, were chosen from the breeding records, that had sired at least 1 litter per week for 4 consecutive weeks.

Where possible at least four piglets from each litter were tested. To reduce the total number of animals to be attested, the litters used in the growth feed conversion study were also used in the heritability study.

The heritability data has been collated for analysis.

There does appear to be some boars whose progeny all have a similar type of adrenal response.

2. Seasonal Variation This study was designed to examine changes in adrenal .2Q, responsiveness between farms, sexes and season. It encompasses 4 farms, located near Bendigo, Shepparton, the La Trobe Valley and at Lillydale in the Outer Melbourne Region. Each farm will be visited once per month for 12 o 0 months when 24 male and 24 female pigs of about 60 kg live S weight will be tested. Date has been collected for 7 months to date but has not been analysed.

3. Breed Differences oI o This study was designed to test whether different breeds of pig under the same management and nutrition differed in their adrenal responsiveness. The breeds tested O. were pure bred Large White, pure bred Landrance, a line with o.oo? at least 80% Duroc and a standard commercial cross breed (of known genetic background). These lines represent the major pig types farnmed in Australia. The results are presentd in table 5. While this data has not been analysed statistically there does not appear to be any difference in the mean response to ACTH challenge, although the range of respoonses may differ with the crossbreds showing a wider 1 21 range in responsiveness.

4. Stress-Related Infertility It is well known that many types of infertility seen in cotrnercial piggeries are caused by or related to a stress response. This study was designed to test whether pigs with a certain type of adrenal responsiveness were predisposed to a particular stress-related infertility known as "Seasonal Infertility" and also to look retrospectively at the life time production of sows in relation to their adrenal response.

Two farms were studied Menangle Farms near Sydney and Mayfair CPC in Bendigo. On both properties approximately 20 sows and 20 gilts were bled and tested each week for 10-12 weeks. The results are summarized in Tables 6 to 9. Once again these results have not been analysed statistically, but several trends show out as being consistent. At Menangle the gilts were tested after mating and from Table 6 it can be seen that the farrowing rate was substantially higher for the animals with a low adrenal response to stress. Similar at Bendigo where th\ gilts were bled prior to mating there were substantial benefits in oUo favour of the low responder, rmore gilts entering the boar o shed attained puberty and were mated, and more of the mated o gilts farrowed. There do not appear to be consistent differences in litter size.

For the sows at both Menangle and Bendigo the differences in reproductive performance appear less clear S cut and are most likely not significant (Table This could be interpreted as meaning that sows once they survive 38a their first litter adapt to the stress of production.

Alternately it may be related to the relatively small sample size.

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4 0 00. 0 0 4 04

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4 P t. 4 4' 0P4 4 4 a 44 *4 4* 4*4 4 4444 4 4 For the Menangle piggery the previous reproductive performance of the sows was analysed retrospectively against their adrenal responsiveness. This analysis is presented for the whole sow population in Table 8 and broken down into parities in Table 9. On the whole population there appear consistent benefits in terms of conception rate, litter size

L

i I- I- UI J 22 and lactational failures for the low responders. When analysed according to their parity it can be seen that the benefits of having a low adrenal response to stress are associated mainly with the Parity 1 sows.

Experiment 6 Metabolic Clearance Rate of Cortisol Experiments were conducted on pre-puberal (18-20 week old) Landrace x Large White gilts (n=12) to establish if differences in adrenal responsiveness between individuals could be explained by differences in the metabolic clearance rate (MCR) or cortisol. Pigs with the highest and lowest cortisol concentration 60 minutes after challenge with ACTH were selected from a pool of 36 commercial pigs. Tritium-labelled cortisol was infused (17-27 ml/hr) continuously for 120 minutes to establish "steady state" conditions. Blood samples (10 ml) were collected at 90, 100, 110 and 120 minutes. Replicate experiments were performed on some pigs. Classification of individual pigs as high or low adrenal responders to ACTH 2,Q challenge was confirmed at the end of the clearance rate 0 0 4 S experiments.

0 '"oo The average MCR or cortisol in the group classed as low adrenal responders was 59.7 7.8 1/hr or 1.01 1/hr/kg 0 o" which was not significantly different from the average 0o MCR in the group classed as high adrenal responders of 60.2 5.9 1/hr or 1.19 1/hr/kg These results suggest that the repeatable differences in adrenal responsiveness to ACTH that exists between individuals within a particular strain of pig depend on differences in the rate of synthesis of cortisol in response o t to ACTH stimulation, rather than on differences in its rate 4 1 of metabolism.

Experiment 7 S Correlation of Adrenal Response to ACTH and Stressors 4 This experiment was designed to correlate adrenal response to ACTH with the animals perception of and response to various stressors. Restraint and transport were used in the first experiment.

23 ,Figure 7 shows the average (10 animals) time course of adrenal response to 15 minutes restraint stress and the response of 4 individual animals. As with adrenal response to ACTH there was a wide variation between individuals in response to restraint stress. From the analysis of variance shown in Table 10 it can be seen that the individual pigs' adrenal response to restraint stress was quite repeatable.

When the adrenal response to restraint was compared to the response to ACTH (areas under curve following stimulus), it re led a high degree of correlation between the two stimuli (Figure A high degree of correlation was also obtained for the comparision of area response to restraint versus concentration at 60 minutes after Synacthen (Figure 9).

The average time course of adrenal response to transport stress is shown in Figure 10. While there was a large degree of variation between individuals, this variation appeared to be random (Table 11). One problem with the transport stress experiments was the way in which the stress could be applied, some animals may not have perceived the stressor and hence responded until the truck actually moved off, while others may have responded as soon as they were taken out of their pens. Thus the variation in 00 Q" time at which the animals actually react to the stressor may .0o 0 account for the poor rtpeatability. This compares to the restraint stress where all animals were snared and stressed within a minute or so of one another.

0C'o °O When the mean response to transport (area) was o compared to the response to ACTH, the correlation co-efficients were 0.516 with ACTH area and 0.533 for 0: concentration at 60 minutes after ACTH.

Table 12 shows the ranking of the mean area response of each animal tested with ACTH, restraint and transport.

I a"\r This data was analysed using a non-parametric test Kendall's Co-efficient of Concordance, the resultant correlation co-efficient was quite significant suggesting that there was in fact a good degree of relationship between how an animal responded to ACTH and how they responded to

UFIIB~

24 stress. Table 12 also shows the ranking of the mean concentration at 60 minutes following ACTH challenge comparing these rankings with the rankings for restraint and transport -reas also gave a significant correlation co-efficient, suggesting that the Dynamic Test was a reasonable measure of stress responsiveness.

0 0 0 0 4 00 6 4 0 0 o A0 0 0 o O 4 *0 0 9 00 90 400 0 0 9 O 0 0 0 0 0 9.9 9f~* 900 0

C

000 0 0 00 00 0 0 0. 00 0 0B* 0 0 ES 0 0 oo- 2 5 TABLE 4 DST GROWTH/FCR TRIAL BENDIGO 1986.

Group Week Cortisol Initial Grower Pre-sale TOTAL Tested Cone. Sucker Wt Wt GAIN (nmol/L) Wt.(kg) (kg) (kg) (kg)

TOTAL

FCR

gm. gain/kg. feed 244.2 252.1 236.8 227.1 240.0 9.2 4.41 3.93 4.62 4.79 4.44 0.32 25.98 25.97 28.48 28.09 27.13 1.16 72.60 79.24 79.43 80.80 46.62 53.26 51.00 52.63 376.5 393.6 324.6 323.3 354.50 31.14 TOTAL GAIN (Female) P 0.023 P 0.0001 Between Weeks Between Groups

MEAN

ST.D

78.02 50.88 3.19 2.59 N.S. Interaction

MEAN

ST.D

443.7 464.4 391.5 391.5 422.8 32.1 692.5 622.5 768.5 663.2 686.7 53.4 A.34 3.95 4.37 4.25 3.98 0.40 3.45 2.99 3.94 4.02 3.60 0.42 23.59 24.73 27.45 26.73 69.20 74.98 79.85 74.40 45.62 50.25 51.85 47.68 25.62 74.61 48.85 1.54 3.77 2.39 341.0 375.7 290.5 272.5 319.92 40.82 341.1 337.2 300.9 279.0 PRE-SALE WEIGHT (Female) P 0.0001 Between Weeks 21.97 21.73 24.89 25.66 66.52 67.00 74.00 70.04 44.55 45.28 48.50 44.58 0.0001 Between Groups N.S. Interaction

MEAN

ST.D

23.56 69.39 45.73 314.54 1.74 2.98 1.63 25.32 lii 0 O* a 0 o 00 0 o Sa a o oo0 00 0 26 TA3LE 4 DST GROWTH/FCR TRIAL BENDIGO 1986- (ContiT.aed) Group Week Cortisol Tested Cone.

(nmol/1) Initial Sucker Wt.(kg) Grower Wt (kg) Pre-sale Wt (kg)

TOTAL

GAIN

(kq) 54.90 55.14 56.95 53 17

TOTAL

FCR

qm. qain/kg. feed 263.8 255.7 240.2 251.6 252.8 8.5 4.34 4.30 4.78 4.64 4.51 0.20 27.55 27.32 27.95 28.95 82.45 82.40 84.90 81.17 416.0 423.4 320.7 326.6 TOTAL GAIN (Male)

MEAN

ST.D

27.94 82.73 55.04 571.69 0.63 1.35 1.34 48.15 P 0.92 P 0.0001 Between Weeks Between Groups 443.5 442.7 400.0 407.6 423.4 195- 3.73 3.49 4.58 4.49 24.92 24.11 27.83 27.00 76.29 76.46 82.17 76.02 51-37 52.35 54.33 49.02 375.4 390.2 311.0 315.0 N-S. Interaction PRE-SALE WEIGHT (Male)

MEAN

ST.D

4.07 25.97 77.73 51.77 347.89 0.47 1.51 2.56 1.91 35.31 P 0-063 673.5 656.6 683.3 698.0 677.9 15.1 3.77 3.35 3.66 4.02 23.89 24.05 24-31 25.57 74.30 74.00 76.60 74.61 50.41 49.95 52.62 49.36 375.8 356.5 302.0 299-0 333.32 33.53 Between Weeks Between Groups P 00001 N.S. Interaction

MEAN

ST.D

3.70 24.45 74.88 50.59 0.24 0.66 1.02 1.23 v4 -27- T, -BLE DST BREED DIFFEREN4CES TRIAL MAYFAIR FARMS BFNDIGO 1986.

Cortisol Coneic nrnol L MEAN Std. Dev.

BREED

RANGE

LANDRACE FEMALE 254 52 158 392 LANDRACE MALE 213 55 Il11 382 X BREED FEMALE 238 96 81 595 X BREED MALE 217 72 121 503 DUROC FEMALE 237 el 52 405 LARGE WHITE FEMALE 246 55 125 409 LARGE WHITE MALE 2,43 54 1 1 365 o 04 4 4 <1 4 4 4 4 0 444 4 4 4 0 4 4 444 4 44 4 4 44 44 44 4 4.4 4 44 4.4

I

4 444.444 4 4 4 .~,4*44 4 4 .44 4 C 444 4 04 4 4 28 TABLE 6 Summer Infertility Trial 1986. GILTS 1. MENANGLE Total Highest Lowest Highest Lowest Population 30% 30% 50% No. Animals 171 50 50 85 86 No. Farrowing 134 35 44 60 74 Farrowing 78.4 70.0 88.0 70.6 86.1 Rate M% Mean Litter Data St.Dev.

Born 8.5 Alive Born 1.9 Dead 1.7 .8 11.6 +-2.7 8.8 .5 2.0 11. 7 +-2.5 8.5 1.7 11.4 2.4 11.5 +-2.9 Litter Weight(kg) 11.5 +-2.7 0 9 004 0 000 0 000 0 0 0 0 0 0006.

00 0 00 0 0 2. B2WDIGO Total Highest Lowest Highest Lowest Population 30% 30% 50% No. Animals 240 72 72 120 120 No. Mated 154 44 51 72 82 Mating 64.2 61.1 70.8 60.0 68.3 Rate No. Farrowing 134 35 46 59 Farrowing 89.3 83.3 93.9 84.3 93.8 Rate

I

-29- TABLE 7 Summer Infertility Trial 1986. SOWS 1. MENANGLE Total Highest Lowest Highest Lowest Population 30% 30% 50% No. Animals 203 60 60 101 102 No. Farrowing 167 50 46 84 83 Farrowing 82.3 83.3 76.7 83.2 81.4 Rate Mean Litter Data St.Dev.

Born 10.0 Alive 2.5 Born Dead 0.6 1 14.4 /-3.4 9.4 .1 0.8 13 .9 +-4.2 3, 0. 1 +-2.5 0.5 14.4 *I-3.4 9.8 6 0.6 14 .3 3.6 10. 1 +-2.4 0.6 14 .6 +-3.2 oa C, o0 0 0. 0 oQo 00 0a 0*0 0 #to Litter Weight (kg) 2. BENDIGO Total Highest Lowest Highest Lowest Population 30% 30% 50% No. Animals 242 74 72 122 120 No, Mated 232 72 68 119 113 Mating 95.9 97.3 94.4 97.5 94.2 Rate No. Farrowing Farrowing Rate 203 89.8 62 88.6 61 92.4 104 89.6 99 90.0 e o 0 0 0 I 1 0 0

LI

a o a~ a 0*0 o o 6a a 30 TABLE 8 DST RETROSPECTIVE REPRODUCTIVE PERFORMANCE STUDY. MENANGLE 1986.

Cortisol Avg. No.

Post Parity Matings

ACTH

Conception Rate Average Litter Data Alive Dead e

N

Wt. Wea Average Total No.

o. Lactational ined V}t. Failures Total Population Highest Lowest Highest No.

Mean St.D No.

Mean St.D No.

Mean St.D No Mean St.D No.

Mean St.D 208 230 9 77.3 62 327.4 63.0 62 158.5 25.9 104 288.1 68.9 104 174.7 28.6 2.4 1.1 2.0 0.8 2.8 1.2 2.2 1.0 2.7 1.3 2.4 1.2 3.1 1,3 2.5 1.2 88.9 83.3 90.3 88.0 9.2 0.6 lz.6 8.1 40.9 1.9 0.8 2.7 1.5 9.1 8.7 0.7 12.2 8.0 40.9 2.0 1.1 2.9 1.4 8.1 9.3 0.7 12.6 8.1 40.0 1.7 0.7 2.1 1.5 7.9 9.1 0.6 12.4 8.0 2.0 1.0 2.8 1.5 40.7 9.3 Lowest 2.6 1.1 92.9 9.4 0.6 12.8 8.2 1.7 0.6 2.6 1.4 41.2 8.9 o 06 0 a o a *i o 0 4 0 0 0 t0 0 -0 e4 aoo o ofto of"J o o o o o o 31- TABLE 9 DST RETROSPECTIVE REPRODUCTIVE PERFORMANCE STUDY. MENANGLE 1986.

ANALYSED BY PARITY PARITY 1 Cortisol Avg. No. Conception Average Average Total No.

Post Parity Matings Rate Litter Data No. Lactational ACTH Alive Dead Wt. Weaned Wt. Failures Total No. 41 2 Population Mean 242.1 1 1.2 83.3 8.8 0.9 11.1 8.0 40.4 St.D 76.2 0.4 2.4 1.2 3.8 1.8 10.1 Highest No. 12 2 Mean 336.8 1 1.3 76.9 7.7 1.0 10.6 7.6 38.2 St.D 57.0 0.5 2.5 1.5 3.8 1.3 Lowest No. 12 0 Mean 167.2 1 1.1 90.9 9.2 0.8 11.5 7.8 40.8 St D 35.7 0.3 2.3 1.0 3.1 1.6 9.2 L~ 99 9 9 9 909 0 32 TABL 9 DST REROPCTV RERDUTV PEFOM£C 0TUDY M L 1986 (Cntnud TALE9 STREROPCTHV REPODUTIV DERORMNC STUDY MENANGL 1986 (Continued Total No. 90 6 Population Mean 245.4 2 2.3 87.0 9.1 0.5 12.6 8.0 41.8 St.D 77.6 0.5 1.8 0.8 2.2 1.4 8.4 Highest No- 27 1 Mean 342.1 2 2.3 87.0 8.5 0.5 12.0 8.2 43.4 St.D 51.6 0.6 1.8 1.2 2.4 1.3 Lowest No. 27 1 Mean 168.0 2 2.2 90.9 9.2 0.5 12.8 8.1 39.7 St.D 24.6 0.4 1.8 0.6 2.0 1.3 .00 S 0 0 P 000 0 -33 TABLE~ 9 DST REROPCTV RERDUTV PEFRAC STDY ME L 196 (Continued) TABLEY 9 DS RETRETIVE REROUCIV PEFoAC Soce tUDY. MEAerge 1986.g (Continued Post Parity Matings Rate Litter Data No. Lactational ACTH Alive Dead Wt. Weaned Wt. Failures Total No- 77 9 Population Mean 207.8 3.6 3.9 92.3 9.6 0.6 13.5 8.2 40.3 St.D 71.8 0.7 1.0 1.5 0.5 2.1 1.4 9.3 Hiqhest No. 23 7 Mean 284.0 3.6 4.0 90.0 10.0 0.4 13.8 7.9 37.8 St-D 82-9 0.6 1.0 1.7 0.3 2.5 1.6 9.8 Lowest No. 231 Mean 146-1 3.7 4.0 92.5 9.2 0.7 12.8 8,.1 41.2 St-D 19.2 0.8 1.1 1.3 0.6 1.5 1.1 6.4

I

Restraint Stress experiments on 10 indivi area under response curve RESTRAINT STRESS RESTRAINT

STRESS~-~-

NO.

1 2 3 4 6 7 8 9 NAME N 4 4 5 4 8 4 9 2 11 2 13 3 15 2 16 2 17 3 20 3

MEAN

1134.2500 1207.2500 1994.0000 857.5000 1455.5000 1519.0000 1189.5000 1555.5000 1052.6667 1348.0000 34 TABLE Analysis. Up to 4 replicate dual pigs. Data is expressed as for the 15 minutes of restraint.

AREA INDIVIDUAL ANIMALS STD. DEV. MINIMUM MAXIMUM 166.6641 947.0000 1323.0000 160.1590 1061.0000 1408.0000 424.3497 1607.0000 2442.0000 28.9914 837.0000 878.0000 91.2168 1391.0000 1520.0000 113.7673 1433.0000 1648.0000 44.5477 1158.0000 1221.0000 21.9203 1540.0000 1571.0000 174.0182 939.0000 1253.0000 96.3172 1271.0000 1456.0000 ANALYSIS OF Header data for: B:AREAIND Label: IND AREA EST Number of cases: 29 Number of variables: 1 ONE-WAY ANOVA 4 94 ooIPDI 4 4 *6 4,00 S94 4 4o t 0 4 t 4 0 4 4 4 3$4 44 1 4* Variable

SOURCE

Between Within SUM OF SQUARES 2920762.040 817132.168

D.F.

9 19 MEAN SQUARE 324529.116 43006.956 ii F RATIO 7.546

PROB.

.0001 Total 3737894.208 28 NONPARAMETRIC Header data for: B:AREAINDR Label: IND AREA EST Number of cases: 29 Number of variables: 1 KRUSKAL-WALLIS TEST H 24.185 D.F. 5 PROB. .0040 35 TABLE 11 Transport Stress Analysis. U- to 3 replicate experiments on 8 individual pigs. Data is expressed as area under response curve the first 30 minutes from commencement of loading. An additional 3 pigs had only 1 replicate of the stress and are not included in the analysis.

TRANSPORT STRESS #1 AREA UNDER CURVE DURING TRANSPORT NO. NAME N MEAN STD, DEV. MINIMUM MAXIMUM 1 4 2 842.5000 183.1407 713.0000 972.0000 2 6 2 1326.5000 214.2534 1175.0000 1478.000 3 8 2 1220.0000 22.6274 1204.0000 1236.0000 4 11 2 1323.0000 557.2001 929.0000 1717.0000 13 3 807.3333 283.0147 636.0000 1134.0000 6 15 3 1194.6667 291.8652 934.0000 1510.0000 7 16 3 1254.3333 207.0201 1049.0000 1463.0000 8 20 2 1229.5000 133.6432 1135.0000 1324.0000 ANALYSIS OF Header data for: B:TAIND Label: o Number of cases: 19 Number of variables: 1 ONE-WAY ANOVA Variable 1: SOURCE SUM OF SQUARES D.F. MEAN SQUARE F RATIO PROB.

0 o Between 722555.132 7 103222.162 1.377 .3048 o Within 824569.501 11 74960.864 Total 1547124.633 18 NONPARAMETRIC 0* Header data for: B:TAIND Label: Number of cases: 19 Number of variables: 1 KRUSKAL-WALLIS TEST S:SO; H 8.121 D.F. 7 PROB. .3220 O* C 4 i I I i 9 o 99r 9 9 Cn *i 69 9c 49 9 96 9 9 99 9 99 9D 9 99 9 #90 0*91 4 9 8 36 TABLE 12 The rankings of the mean response of individual animals following challenge with ACTH, ranked according to means cortiol concentration at 60 minutes and mean area under curve; and following restraint and transport stress ranked according to mean area under curve.

Animal ACTH Restraint Transport No. 60 minutes Area Area Area 1 3 2 3 2 9 7 4 3 6 3 6 4 14 10 13 13 12 6 6 10 11 11 9 7 1 1 1 1 8 8 6 8 12 9 7 5 9 4 10 5 4 5 8 11 11 9 10 11 12 2 2 3 7 13 4 2 14 12 8 7 1. Kendall's Co-efficient of Concordance for ACTH area Vs Restraint Area Vs Transport Area r 0.644 P 0.0194 2. Kendall's Co-efficient of Concordance for ACTH minutes Vs Restraint Area Vs Transport Area r 0.567 P 0.0294 -37 Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

0 64 a a 06 66 a 00 66

Claims (11)

1. A method for testing the reaction of animals to stress-related stimuli which method includes: providing an animal to be tested, and a predetermined amount of an adrenal stimulant; administering the adrenal stimulant to -he animal; and measuring the adrenal response after a predetermined interval of time.

2. A method according to claim 1 wherein the adrenal stimulant is an adrenocorticotrophic hormone.

3. A method according to claim 1 wherein the animal is porcine.

4. A method according to any one of claims 1 to 3 wherein the adrenal stimulant is 25 to 50 iu of synthetic ACTH and the interval of time is 50 to 70 minutes post injection. A method according to claim 1 wherein the adrenal stimulant is selected from Tetracosactrin or Synacthen,

6. A method according to any one of 1 to 5 wherein the adrenal response is measured by measurement of the corticosteroid level in a blood sample, 04

7. A method according to claim 6 wherein the measurement of the corticosteroid level is measured by a radioimmunoassay method.

8. A method according to claim 7 wherein a radioactive cortisol, 125 I-labelled cortisol is used in a double antibody radioimmunoassay. S9. A method according to claim 6 wherein the measurement Sof the corticosteriod level is measured by an ELISA said method including use of; i) an antibody against cortisol ii) an enzyme-linked antibody conjugate and iii) an enzyme substrate. A method according to claim 9 wherein the antibody is generated by immunising an animal with a cortisol:protein conjugate.

11. A method according to claim 10 wherein the cortisol:protei, conjugate is a cortisol 3-methyl oxime:bovine allumine conjugate. 39

12. A method according to any one of claims 9 to 11 wherein the enzyme-linked anriibody is selected from alkaline phosphates, glucose oxidase, a horseradish peroxidase or urease.

13. A method according to any one of claims 9 to 12 wherein the enzyme substrate i t'eiacted from an ABTS, an 0-toludine solution or a standard urease substrate solution.

14. A method according to claim 1 substantially as hereinbefore described. A method for identifying in a population of animals those individuals which exhibit a low response to stress related stimuli, which nethod includes: providinq a plurality of animals and subjecting said animals to a method of stress-testing as defined by any one of claims 1 to 14; and selecting animals exhibiting a low adrenal response for a selected breeding program, 16, A diagnostic test kit/ for conducting a method as defined by any one of claims 9 to 13, said kit including: a vessel suitable for in an enzyme-linked immuno absorbent assay; an antibody against cortisol in a suitable continer; an enzyme-linked antibody conjugate in a suitable s container; and San enzyme substrate in a suitable container. 17, A diagnostic test kit according to claim 16 wherein i o the vessel is a microtitve plate. DATED: 10 July 1991 PHILLIPS ORMONDE FITZPATRIC;" Attorneys For: CIBA-GEIGY AUSTRALIA LIMITED 3017h