Clinical signs, findings of the physical examination and results of further diagnostic methods confirmed the diagnosis of DI in two Duroc boars originating from a semen collection centre. There are several forms of DI described, but in the present study it was not possible to classify the type of DI accurately, as there are some limitations in diagnostics for pigs.
With a clinical history characterized by polyuria and polydipsia in pigs several differential diagnoses have to be kept in mind and each one has to be excluded from the list of differential diagnosis. Therefore, clinical examination and further tests are essential and need to be performed sequentially. During the physical examination the urinary bladder of both boars were extremely full and distended, but dehydration, which is often reported in DI [19], could not be observed. It is noteworthy that in pigs there is no adequate method to measure the hydration status and therefore experimental studies in DI use diuretic medication [20] or fluid restricted pigs [21] for evaluation. As a next step, analysis of the urine was performed and revealed a hyposthenuria with a specific gravity of 1.001 in one boar and 1.002 in the other boar. The specific gravity in pigs, which is the lowest among animals, usually is 1.020 on average and can range between 1.010 and 1.050 [22, 23]. No further abnormalities such as glucosuria or inflammatory signs were detected. Several blood compounds are able to provoke dysfunction of the renal system, hence a complete blood count was conducted and showed a slight chronic inflammation, which might have been caused by the pneumonia.
As a further diagnostic approach a modified water deprivation test was performed. The test is designed to determine, whether ADH is released in response to dehydration and if the kidneys are able to respond normally to the hormone [1–3]. The aim of the test is to achieve maximal ADH secretion and thereby higher concentrated urine. This commonly occurs after a 3 to 5 % loss of body weight due to water loss, which makes it necessary to measure the body weight several times. Additionally, emptying of the bladder with a catheter is required [1]. Due to the characteristic anatomy of the urethra of male pigs, transurethral catheterization of the bladder is impossible [24]. The endpoint of the water deprivation test is a loss of body weight greater than 5 % or a specific gravity of the urine higher than 1.030 [1]. A more accurate measurement is the assessment of the urine’s osmolality [1]. Unfortunately, this was not feasible throughout the whole water deprivation test, because of logistical and financial reasons. The osmolality of urine can also be approximated by multiplying the last two digits of the urine specific gravity by 36 [25]. There was one single measurement of urine’s osmolality of case #2, which was performed during the trial and revealed a result of 32.5 mOsm/Kg. Comparing the measured value with the calculated urine osmolality of 36 mOsm/Kg, there was a good correlation, which can also be observed in other species, e.g. in dogs.
The pig is a good model for renal research. The ratio between urine and serum osmolality in healthy pigs and healthy humans is 3.3 [21]. In the literature, there are numerous different equations described to calculate serum osmolarity in humans. One group reported an equation (Eq. 1) derived from results of an experiment in pigs, where concentrations are expressed in mEq/L [26].
Equation 1:
$$ \mathrm{Serum}\ \mathrm{osmolarity}\ \left(\mathrm{mEq}/\mathrm{l}\right) = 1.8177\ *\ \left[\mathrm{N}\mathrm{a}\right]\ \left(\mathrm{mEq}/\mathrm{L}\right) + \left[\mathrm{Urea}\right]\ \left(\mathrm{mEq}/\mathrm{L}\right) + \left[\mathrm{Glucose}\right]\ \left(\mathrm{mEq}/\mathrm{L}\right) + 26.05 $$
A baseline of 294.9 with a SD of ± 1.8 mOsm/Kg in 10–40 kg femal Yorkshire-Duroc crossbred pigs was reported [27]. Furthermore, in fattening pigs 284.74 ± 5.73 mOsm/Kg [26] was described. In order to get an indication for serum osmolarity in boars, additional three Duroc boars at the age of 8 month to 1.1 year from the semen collecting centre were tested. A reference range from 321 to 326 was measured. The calculated value of serum osmolarity in case #2 was 290.9 mOsm/Kg and, thus, within the above mentioned reference values taken from the literature. In contrast, the calculated value was significantly lower than those measured in the control boars. However, the result generally must be interpreted with caution, because there is a significant bias by food and water intake [28], which can have an impact on the level of osmolarity. If we compare the ratio of serum osmolarity with urine osmolality it is almost three times higher than the average value of healthy pigs.
After the water restriction test a therapeutic attempt was tried, as it is commonly performed with small animals and also with horses. The animals are treated with desmopressin acetate (DDAVP), a synthetic analogue of ADH. There are several routes of administration available, but most often the conjunctival route is chosen. Therefore, one to two drops are applied into the conjunctival sac of both eyes every 12 to 24 h [3, 29]. The eye drop method is a non-invasive, practical and effective way of hormone administration [30]. An oral application of desmopressin is possible, but the bioavailability is lower compared to the afore mentioned method [19, 31]. The duration of the effect of DDAVP varies from eight to 24 h [3]. Since no specific dosage regime was available for pigs, a treatment of the boar 2-3 times per day with 3–5 drops in the conjunctival sac of one eye was proposed. Only a slight decrease of the urine specific gravity was observed. In small animals this result would lead to diagnosis of primary NDI. However, the porcine vasopressin contains a lysine residue in position 8 (lysine-vasopressin), which makes the pig quite different from other mammalian species, where vasopressin contains an arginine residue in position 8 (arginine-vasopressin) [9]. Therefore the porcine V2 receptor has less sensitivity to desmopressin than human V2 receptors. In the literature a two hundred times lower affinity of desmopressin on porcine V2 receptor is described [32]. Even in high doses desmopressin did not induce any hematological response [33]. The authors clinical interpretation is that desmopressin cannot be recommended for diagnostics or treatment in pigs due to the implications mentioned above.
Unfortunately, we performed both diagnostic approaches, therapeutic attempt and modified water deprivation test, only in one boar, because it was only possible to keep one boar in our facilities, regarding to Swiss legislation. To prove the efficiency for both tests in a larger number of pigs with DI, warrants further investigation.
Another diagnostic tool for the confirmation of the disease’s aetiology is the measurement of endogenous ADH in plasma, where in case of diseases osmotic and cardiovascular homeostasis are disturbed [4]. However, the reliability of assessments of plasma ADH levels is poor because the hormone is unstable, largely attached to platelets, and rapidly cleared from plasma [34, 35]. Therefore, the level of a precursor of ADH, Copeptin, which is stable for days, is usually measured in plasma samples [35]. Test kits, specifically designed for pigs, are available for the purpose of clinical research, but they are rarely used and in the presented case the authors could not find a laboratory offering the determination of Copeptin in swine plasma.
The genetic analysis revealed that all the candidate variants in the three DI candidate genes were obviously not associated with the disease and therefore no genetic explanation of the phenotype was found by sequencing of these three genes. Pedigree analysis showed a large number of common ancestors among the affected cases indicating inbreeding. But these shared ancestors are some generations ago which decreases the likelihood of a common inherited simple recessive mutation. Nevertheless, a dominantly inherited mutation with a late onset of clinical signs could also be the cause of the disease, as for example human CDI is caused by dominant mutations of AVP [14]. A recessive inheritance analogue to AQP2 mutations in humans [10] with inbreeding loops further behind in the pedigree is possible as well as an X-linked disease like NDI caused by AVPR2 mutations in humans [14]. Even though, both types of mutations usually manifest in the first weeks after birth in humans. As the phenotype in the boars was recognized later in life one can also hypothesize that a dominant mutation in one of these three genes which is located in the upstream, intronic, or downstream regions affecting the expression level is causing the disease. The used approach is only appropriate to detect variants in the coding region and therefore other more comprehensive methods like genome-wide-association mapping in combination with sequencing of the genome of one case could be useful to map the responsible locus in the swine genome and finally to find the causative mutation.