Abstract: Six pony mares deprived of water for 24 hours showed significant increases in plasma vasopressin (2.8 pg/ml) and osmolality (9 mosmol/kg). When water was made available the ponies drank rapidly (5 of 6 drank to satiety within 90 seconds) and corrected their fluid deficits precisely. Vasopressin did not return to predehydration levels until osmolality did after 15 minutes of access to water. The horse differs from rodents and humans, but is similar to pigs in that vasopressin levels do not fall before osmolality returns to normal. Oropharyngeal factors, therefore, may not be as important in vasopressin release in horses as in other species.

Abstract: REASONS FOR PERFORMING STUDY: Dehydration and heat stress are serious welfare issues for equids working in developing countries. There is a lack of any standardised method or validated interpretation of the skin tent test in horses and donkeys. Owners of dehydrated and heat-stressed animals often depend on veterinary examination for identification of these conditions, leading to delays in treatment and unnecessary reliance on external sources to effect welfare improvement. OBJECTIVES: To validate a standardised skin tent test for dehydration and a behavioural measure of heat stress in working equids; and to examine the effect of heat stress and dehydration on tripping and staggering behaviour. METHODS: The study was carried out on 130 working horses and donkeys in Pakistan. Associations between skin tent and blood parameters (packed cell volume [PCV], serum total protein [TP], serum osmolality), clinical parameters, resting and drinking behaviour were examined. Heat stress behaviour (increased respiratory rate and depth, head nodding, flared nostrils, apathy) was observed in conjunction with rectal temperature. Tripping and staggering were assessed using a simple obstacle course. RESULTS: In both species, heat stress behaviour was significantly associated with increased rectal temperature (P<0.001). A positive skin tent test was not significantly associated with PCV or TP, although in donkeys it was significantly associated with lower serum osmolality (P<0.001). More animals age >15 years had a positive skin tent than those in younger age groups (P = 0.037). Very thin horses were more likely to have a positive skin tent than those in thin or moderate condition (P = 0.028). There was no significant correlation between skin tent and tripping or staggering in either species. CONCLUSIONS AND POTENTIAL RELEVANCE: Heat stress behaviour is related to increased body temperature in working horses and donkeys. Owners may use this to make judgements regarding rest and cooling, precluding the need to seek veterinary attention. The skin tent test for dehydration used in this study did not show a significant relationship with PCV or TP. However, the use of blood parameters to validate the skin tent test may be confounded by anaemia, hypoproteinaemia or electrolyte depletion. Alternative methods are needed to confirm or refute the validity of the skin tent test in working equids.

Abstract: Six pregnant mares were used to determine what level of water restriction causes physiological and/or behavioural changes indicative of stress. Nonlegume hay was fed ad libitum. During the first week of restriction, 5 l water/100 kg bwt was available, during the second week 4 l/100 kg bwt and, during the third week, 3 l/100 kg bwt. Ad libitum water intake was 6.9 l/100 kg bwt; at 3 l/100 kg bwt water intake was 42% of this. Daily hay intake fell significantly with increasing water restriction from 12.9 +/- 0.75 kg to 8.3 +/- 0.54 kg; bodyweight fell significantly for a total loss of 48.5 +/- 8.3 kg in 3 weeks. Daily blood samples were analysed; osmolality rose significantly with increasing water restriction from 282 +/- 0.7 mosmols/kg to 293.3 +/- 0.8 mosmols/kg bwt, but plasma protein and PCV did not change significantly. Cortisol concentrations fell from 8.1 ng/ml to 6.4 ng/ml over the 3 week period. Aldosterone fell from 211.3 +/- 74.2 pg/ml to 92.5 +/- 27.5 pg/ml at the end of the first week. The behaviour of 4 of the 6 mares was recorded 24 h/day for the duration of the study. The only significant difference was in time spent eating, which decreased with increasing water restriction from 46 +/- 3% to 30 +/- 3%. It is concluded that water restriction to 4 l/100 kg bwt dehydrates pregnant mares and may diminish their welfare, but is not life- or pregnancy-threatening.

Abstract: An ultrasonic flow probe was implanted around a branch of the left renal artery in five horses. The effects of dopamine were studied in the unsedated horses 10 days after surgery. Three experiments, separated by at least two days, were performed in random order on each horse. In two experiments, dopamine was infused intravenously for 60 mins at either 2.5 and 5.0 micrograms/kg bodyweight (bwt)/min. Saline was infused for 60 mins before and after each infusion, and for 180 mins in the third experiment as a control. Renal blood flow increased during administration of dopamine at both dose rates (P = 0.0001). Urine volume increased (P = 0.055), and osmolality decreased (P < 0.05), with infusion of dopamine at 5.0 micrograms/kg bwt/min. Arterial blood pressure and heart rate were not significantly affected. Fractional excretions of sodium and potassium were not significantly changed with dopamine infusion. The higher dopamine dose rate was accompanied by dysrhythmias in some horses.

Abstract: The partition of human antibodies in aqueous two-phase systems (ATPSs) of polyethylene glycol (PEG) and phosphate was systematically studied using first pure proteins systems and then an artificial mixture of proteins containing 1mg/ml human immunoglobulin G (IgG), 10mg/ml serum albumin and 2mg/ml myoglobin. Preliminary results obtained using pure proteins systems indicated that the PEG molecular weight and concentration, the pH value and the salts concentration had a pronounced effect on the partitioning behaviour of all proteins. For high ionic strengths and pH values higher than the isoelectric point (pI) of the contaminant proteins, IgG could be selectively recovered on the top phase. According to these results, a face centred composite design was performed in order to optimise the purification of IgG from the mixture of proteins. The optimal conditions for the isolation of IgG were observed for high concentrations of NaCl and low concentrations of both phase forming components. The best purification was achieved using an ATPS containing 8% (w/w) PEG 3350, 10% (w/w) phosphate pH 6 and 15% (w/w) NaCl. A recovery yield of 101+/-7%, a purity of 99+/-0% and a yield of native IgG of 97+/-4% were obtained. Back extraction studies of IgG to a new phosphate phase were performed and higher yields were obtained using 10% phosphate buffer at pH 6. The total extraction yield was 76% and the purity 100%.