The effect of infection with the abomasal nematode, Haemonchus contortus, on the avoidance behaviour of sheep in a motivational-choice test.

The behaviour of immune and non-immune sheep infected with H. contortus and undergoing a variety of experimental treatments was investigated in a motivational-choice test, the arena test. This test evaluates motivational state in sheep by pitting the motivation of test animals to approach a small flock of sheep against the motivation to avoid a human decoy located directly in front of the small flock. Approach distance is decreased by infection in immune ewes but was unaffected by infection in non-immune weaner lambs in the present study. Experimental drug-treatments with the opiate-antagonist nalorphine, the antihistamine chlorpheniramine, and the immunosuppressive glucocorticoid dexamethasone, affected avoidance behaviour but did not shed light on possible mechanisms involved in the changes observed when immune sheep are infected with the parasite. These substances may affect motivational state directly and not through a pharmacological action on processes triggered by infection in immune sheep. Arena tests conducted in immune ewes at 4, 7 and 11 days after challenge infection showed a fluctuating locomotor behaviour, which may arise from either the dynamics of a standard secondary immune response or particular antigens released during larval development. The immune-mediated changes in behaviour in the arena test will have entailed information-processing or cognitive pathways, but it is not known whether they also involved the physiological manifestations of emotion.

Effects of ovine corticotropin-releasing factor and vasopressin on plasma beta-endorphin, cortisol and behavior after minor surgery in sheep.

The evidence for an analgesic effect arising from increased peripheral concentrations of beta-endorphin (beta-EP) in various animal species is controversial, and has not been fully evaluated in the sheep. To stimulate beta-EP release, ovine corticotropin-releasing factor (oCRF) and arginine vasopressin (AVP) were injected intravenously (iv) into a group of 12 out of 24 sheep, 15 min prior to minor surgery on all sheep. This brought about significant increases (P less than 0.01) in plasma beta-endorphin (beta-EP) and cortisol concentrations, relative to the non-injected control sheep, 15-30 min after injection. Ultrafiltration indicated that less than 30% of the released beta-EP immunoreactivity was present as higher molecular weight forms (mol. wt greater than 10,000) and that the majority (about 75%) of the beta-EP was probably bound to plasma proteins. By 75 min after injection there was no significant difference in plasma beta-EP or cortisol concentrations between the two groups of sheep. Consistent with previous observations the sheep showed a characteristic aversive behavior to the human handler following surgery, lasting several days. This behavior appeared to be unaffected by a pre-operative increase in peripheral plasma beta-EP, and may indicate that this increase in beta-EP was not sufficiently analgesic to block the cognitive response to the operation, or long-lasting enough to prevent the perception of post-operative soreness.

Stress in calves castrated surgically or by the application of rubber rings.

The effects of castration were studied in calves 4 to 11 weeks of age, using increases in salivary cortisol as an indicator of stress. Groups were castrated surgically or by rubber ring application or were non-castrated (control) females. The surgically castrated group showed more agitation during the operation, but both castrated groups resumed normal behaviour soon after the operation was completed. The short-term salivary cortisol response was significantly higher after surgical castration than after the application of rubber rings, where, in turn, it was significantly higher than in the control group. Salivary cortisol was elevated over a period from 15 min to 3 h following the castration, but at 4, 24 h and 6 days post-treatment there were no significant differences between treated groups and controls.

Stress responses in lambs docked and castrated surgically or by the application of rubber rings.

A comparative study has been made in lambs 3 to 6 weeks of age of the behavioural responses and changes in plasma immunoreactive beta-endorphin (ir beta-endorphin) and cortisol after docking or docking plus castration by the application of rubber rings or by surgery. The use of rubber rings on lambs at this age was characterised by very agitated behaviour indicative of considerable distress for a period of up to 1 h. In contrast, surgery was accompanied by some post-operative shock exhibited by reduced exploratory and social behaviour, at least in the lambs exposed to docking plus castration. In the latter group there were highly significant increases in both plasma ir beta-endorphin and cortisol concentrations that may be consistent with the induction of stress-induced analgesia. We conclude that surgery caused less distress than the rubber rings, and is therefore preferable for docking and castration of lambs 3 to 6 weeks of age.

Development of a salivary cortisol method for detecting changes in plasma "free" cortisol arising from acute stress in sheep.

A simple device for collecting saliva (mainly parotid) from sheep is described. The collection of saliva, and the assay of "free" cortisol in saliva appears to offer certain advantages over the collection of blood, and the assay of serum cortisol, for the assessment of stress in sheep. With a little experience, it is easier to collect saliva than take blood samples when sheep are passing through a race. The "free" cortisol can be measured directly in saliva, whereas in serum, it is first necessary to separate "free" from protein-bound cortisol. Basal levels of "free" cortisol of less than 10 nmol/l were recorded in saliva and blood plasma or serum in unstressed sheep which had previous experience of being handled in a race, Significant increases in salivary cortisol and "free" and total ("free" plus protein-bound) cortisol in serum were found in sheep following adrenal stimulation with synacthen, or after 30 min of stressful transport. This indicates that the salivary cortisol technique is applicable to studies of stress in sheep, and should also be useful for other ruminants.

A comparison of stress in surgically and non-surgically mulesed sheep.

A comparison has been made in 9- to 10-month-old castrated male Merino sheep of the changes in plasma total cortisol concentration and behaviour after being treated by either the modified Mules operation or by topical application of a quaternary ammonium compound to achieve non-surgical mulesing. After surgical mulesing, plasma total cortisol concentration increased immediately and rapidly and reached a peak value in 15 minutes, whereas after non-surgical treatment an immediate rise did not occur, but a similar peak value was observed in blood samples collected 24 hours after treatment. The concentrations were lower in both groups at 48 hours. Likewise postural changes indicative of discomfort were immediately apparent in the surgically treated sheep, but not until 3 to 4 hours later in those treated non-surgically. Arena testing revealed that a lasting aversion to the person who restrained them during treatment developed in the surgically mulesed sheep, but not in those treated non-surgically. The non-surgical procedure did not create large open wounds, as did the surgical operation, but still achieved similar enlargement of the bare area on the breech, and healing was quicker in the non-surgically treated sheep.

The intensity of resistance by mature Merino ewes against Haemonchus contortus and Trichostrongylus colubriformis in single-species and combined-species infection.

Three-year-old, non-lactating and non-pregnant Merino ewes, raised on pasture under a program of strategic treatment with anthelmintic and found to be extremely resistant to "trickle" infection with Haemonchus contortus, were given single-dose infections with either H. contortus or Trichostrongylus colubriformis or both species together. The purpose was to ascertain the intensity of protective immunity against the 2 parasites in sheep with immunity acquired from a presumably slight exposure to infection. To provide a criterion, some infected ewes were immunosuppressed with corticosteroid, dexamethasone. Untreated ewes were extremely resistant to challenge infection with either 15,000 or 150,000 H. contortus or 15,000 T. colubriformis. Surprisingly, when mixed infection was given, egg counts for H. contortus were significantly elevated compared with infection by that species alone. Antibody to antigens from infective larval and adult H. contortus was measured in serum by enzyme-linked-immunosorbent assay (ELISA) during the course of infection. Serum titres against larval antigens were significantly depressed when infections with either H. contortus or T. colubriformis were permitted by immunosuppression with dexamethasone, whereas those against adult antigen were depressed when infection with T. colubriformis was permitted.

Effect of ACTH and oxytocin treatment on lactoferrin and citrate in cows' milk.

Six dairy cows were treated before milkings with either oxytocin (Pitocin, 20 i.u.) or ACTH (Synacthen, 150 i.u.), principally to determine their effect on the ratio of citrate: lactoferrin concentrations in the milk. With ACTH treatment, after 3 d milk yield and citrate concentration decreased significantly, lactoferrin and bovine serum albumin (BSA) concentrations increased significantly. Somatic cell counts (SCC) increased temporarily in the milk of three of the cows which previously had greater than 100 000 cells/ml. Lactoferrin yield remained fairly constant but citrate yield was significantly reduced. The citrate: lactoferrin molar ratio decreased from 1373 to 606. With oxytocin treatment, after 4 d milk yield first increased and then significantly decreased, citrate concentration decreased significantly while there were no significant changes in lactoferrin or BSA concentration or in the yield of any other milk constituents. The citrate: lactoferrin molar ratio decreased from 1621 to 1301. There were no significant changes in SCC either during treatment or 4 d after treatment but there was a significant rise at 16 d after treatment. It was concluded that in lactating cows both hormones affected citrate and lactoferrin concentrations in the direction that would improve the antibacterial properties of milk, but that this was accompanied by adverse effects on milk secretion. The extent of the change was not sufficient to be likely to produce inhibition of coliform bacteria.

Comparison of total and free cortisol in bovine serum and milk or colostrum.

Total (free plus protein-bound) and free cortisol concentrations in paired samples of serum and skim milk or colostrum were determined by specific radioimmunoassay after extraction or ultrafiltration. For 11 cows in established lactation mean total cortisol concentrations +/- SE in serum and milk were 4.5 +/- .7 ng/ml and .35 +/- .06 ng/ml, respectively. Concentrations of free cortisol in serum and milk were about .3 ng/ml, which is at the lower limit of the present assay. Following 200 IU Synacthen injection to 5 cows, total cortisol in serum and milk rose to 67 +/- 6.0 ng/ml and 11.4 +/- 1.5 ng/ml by 8 h postinjection, while free concentrations increased to 4.3 +/- .7 ng/ml and 6.5 +/- .8 ng/ml in serum and milk, respectively. Eight additional cows were sampled at 1 to 2 d after parturition, and total cortisol in serum and colostrum was 16.6 +/- 1.6 ng/ml and 4.4 +/- 1.3 ng/ml, and free cortisol was 2.4 +/- .4 ng/ml and 1.8 +/- .5 ng/ml, respectively. Free cortisol concentrations in colostrum or in milk from cows in established lactation, before or after adrenal stimulation, are directly related to free cortisol concentrations in the blood, while the bound cortisol in milk is more related to these two stages of lactation rather than the bound hormone in the blood. Measurement of free cortisol in milk should allow the monitoring of changes in plasma free cortisol in studies of stress in dairy cows.

Changes in behaviour, cortisol and lymphocyte types during isolation and group confinement of sheep.

This experiment compared changes in complex behaviour patterns, adrenal corticosteroid secretion and the numbers of various types of lymphocytes in sheep that were subjected to the stress of confinement. Grazing Merino ewes (n=80 in five replicated experiments) were confined either in groups of four per pen or in total isolation from other sheep. The percentage of CD4+ lymphocytes increased while the percentage of CD8+ lymphocytes decreased over the experimental period. This result was more pronounced in isolated sheep than in grouped sheep. The increase in CD4:CD8 was greater for isolated sheep than for grouped sheep and greater for 2 week sheep than for 3 week sheep. The percentage of CD5+ cells also increased, less so in isolated than in grouped animals. Interpreting these changes as a recovery of immune competence following introduction of a stressor, it is apparent that isolation impaired immune system recovery more severely than group confinement. Physiological and behavioural adaptation over the period were characterized by a decline in the adrenocortical response, resumption of the normal pattern of flocking behaviour and a reduction in motor activity during the test. These findings add to the evidence pointing to the possible correspondence between critical features of the psychoneural, neuroendocrine and immune systems.

Determination of staphylococcal enterotoxin A in cheddar cheese produced without starter activity.

Three variants of the chloramine-T radioiodination method were used to iodinate staphylococcal enterotoxin A with 125I. Only one method consistently produced usable labels for radioimmunoassay. The iodine incorporation was 55 to 76%; the specific activity was 3.5 to 5.5 muCi/microgram of enterotoxin, and the label was extremely stable on storage at -20 degrees C. Determinations of the enterotoxin in extracts of cheddar cheese produced without starter activity were carried out with the radioimmunoassay system and protein A as antibody immunoadsorbent. The assay buffer used in this system significantly influenced the detected levels of enterotoxin in the cheese extracts. Phosphate buffer, but not tris(hydroxymethyl)aminomethane (Tris) buffer, caused gelling of cheese extract proteins, thus resulting in an incomplete separation of free from antibody-bound 125I enterotoxin. When Tris buffer was used, the results indicated a high degree of accuracy and precision for this radioimmunoassay. The lowest detectable enterotoxin concentration in cheese extract was 0.5 ng/ml.

The effect of 2-bromo-alpha-ergocryptine and 2-chloro-alpha-methylergoline-8beta-acetonitrile (lergotrile mesylate) on the prolactin secretion of the ewe.

The effects of two ergot alkaloids, 2-bromo-alpha-ergocryptine methane-sulphonate (CB154, Sandoz) and lergotrile mesylate (LM, E. Lilly & Co.), on the basal secretion of prolactin and on the prolactin response to TRH or the milking stimulus was investigated in the sheep. Both CB154 (c. 0.5 mg/kg) and LM (c. 0.75 mg/kg) markedly reduced basal levels of prolactin and inhibited the TRH-induced prolactin release in ewes in the mid-luteal phase of the oestrous cycle. Both compounds also suppressed basal levels of prolactin in lactating ewes and inhibited the prolactin response to the milking stimulus in lactating ewes.

Artificial induction of lactation in ewes: the role of prolactin.

The mammary glands of 30 non-pregnant, intact ewes were developed by subcutaneously injecting oestrogen plus progesterone at intervals of 3 days from day 0 to day 27. Two days later (day 29), 15 ewes were injected subcutaneously with 18 mg ergocryptine, to inhibit specifically secretion of prolactin. Then groups of ewes, each comprising five ergocryptiine-treated and five untreated ewes, were injected from days 30 to 34 with either four intravenous injections each day of 1 i.u. syntocinon, one subcutaneous injection each day of 10 mg dexamethasone trimethylacetate, or two subcutaneous injections each day of 2-5 mg oestradiol benzoate plus 6-25 mg progesterone. All ewes were milked by hand on days 30-50. Within 24 h of injecting ergocryptine, levels of prolactin in serum were reduced to negligible values (less than 2 ng/ml). Comparison of results for ewes not receiving ergocryptine showed that syntocinon, dexamethasone and oestradiol benzoate plus progesterone, at the doses used, were equally effective in initiating milk secretion. Peak yields of 0-23-0-27 kg/day were achieved. On the other hand, ewes treated with ergocryptine before syntocinon or dexamethasone produced peak yields of only 0-12-0-13 kg/day and ewes treated with ergocryptine before oestradiol benzoate plus progesterone produced negligible amounts of secretion. The results suggest that syntocinon and dexamethasone were either lactogenic per se or effected the release of hormones of the lactogenic complex other than prolactin. However, oestradiol benzoate plus progesterone appeared to be lactogenic by virtue of the influence of oestrogen on the secretion of prolactin.

Effect of myiasis and acute restraint stress on plasma levels of immunoreactive beta-endorphin, adrenocorticotrophin (ACTH) and cortisol in the sheep.

Cutaneous myiasis in sheep arising from the activity of Lucilia cuprina larvae can result in significant physiological changes in susceptible animals. The stress imposed on the pituitary-adrenal axis of the sheep in response to myiasis and acute restraint is the subject of this investigation. Merino wethers were exposed to handling restraint, and blood sampling, during examination for blowfly strike; where necessary, they were treated for cutaneous myiasis. Significant changes in the plasma concentrations of immunoreactive beta-endorphin (beta-EP), ACTH and cortisol were found in sheep with extensive myiasis, as compared with unstruck sheep or those with only localized myiasis. In five susceptible sheep with extensive cutaneous myiasis, mean plasma levels of beta-EP, ACTH and cortisol were 307 +/- 71 pg ml-1, 953 +/- 58 pg ml-1 and 232 +/- 46 nmol l-1 respectively, compared with 818 +/- 89 pg ml-1, 641 +/- 41 pg ml-1 and 107 +/- 17 nmol l-1 in six unstruck sheep handled similarly. Whereas significant increases in plasma ACTH and cortisol can result from pituitary-adrenal responses to acute emotional or surgical stress, and are usually accompanied by a concomitant release of beta-EP from the pituitary, the present findings indicate a marked reduction in beta-EP levels and a significant increase in ACTH and cortisol in sheep following blowfly strike and acute handling restraint. This result suggests that cutaneous myiasis in susceptible sheep can alter the pituitary-adrenal response to acute restraint stress, and this could occur either by an alteration of precursor processing in the pituitary or by the selective release of ACTH.

Artificial induction of lactation in ewes: the involvement of progesterone and prolactin in lactogenesis.

An attempt has been made to evaluate the importance of prolactin and 'progesterone withdrawal' for lactogenesis. The experimental model system used was the ovariectomized, non-pregnant ewe induced to lactate artifically by treatment with trigger hormone (either oestrogen, glucocorticoid or oxytocin) alone or in combination with progesterone. It appears from the results that prolactin is important in the lactogenic responses elicited by oestrogen and oxytocin but not as important in the response elicited by glucocorticoid. Moreover, the results suggest that, in the ewe, an appropriate positive hormonal stimulus will overcome the inhibitory influence of progesterone on lactogenesis.

Artificial induction of lactation in ewes: the use of prostaglandin.

Injections of an anlogue of prostaglandin F2alpha (T.F.101) initiated secretion of copious amounts of fluid resembling normal ovine milk when given to non-pregnant ewes with developed mammary glands. Injections of T.F.101 elicited a substantial but transient increase in the levels of prolactin in plasma. Results for intact and ovariectomized ewes were similar.

Stress-induced changes in plasma concentrations of immunoreactive beta-endorphin and cortisol in response to routine surgical procedures in lambs.

Following four different surgical procedures in lambs 3-5 weeks old, plasma immunoreactive beta-endorphin (beta-EP) and cortisol were assayed at 15 min and 24 h as determinants of post-operative stress. A threefold increase in mean plasma beta-EP levels occurred 15 min after tail docking, and a maximal eight- to tenfold increase occurred in response to castration and/or mulesing with tail docking. Significant increments in mean plasma cortisol levels followed these surgical procedures with the maximal response 15 min after mulesing plus castration with tail docking. The physiologically active 'free' cortisol in plasma represents about 25% of the cortisol, as measured, and the two are highly correlated. At 24 h, beta-EP levels in all treated groups were similar to controls, although a small elevation in cortisol levels was still present in the lambs subjected to mulesing. Ultrafiltration of plasma extracts showed that peak beta-EP levels contained about 40% immunoreactivity from low molecular weight species (mol. wt less than 10,000). By specific radioimmunoassay and reverse-phase high-performance liquid chromatography this comprised about 75% beta-EP1-31, the most potent analgesic endorphin, 10% beta-EP1-27, and 15% alpha-N-acetyl-beta-EP. Increased beta-EP1-31 levels may modulate post-operative pain in lambs.