RENAL UREA EXCRETION IN SINGLE- AND TWIN-PREGNANT GOATS

Katarzyna Michałek, Małgorzata Ożgo, Wiesław F. Skrzypczak
Departament of Animal Physiology and Cytobiology, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology, Szczecin, Poland

ABSTRACT

The aim of the studies was to trace changes in urea blood plasma concentration and renal excretion and to compare these between groups of single- and twin-pregnant goats. Clearance tests involved 16 pregnant White Improved does (8 in single- and 8 in twin pregnancy). Urea concentration was determined in blood plasma and urine. It has been demonstrated that renal nitrogen metabolism in pregnant goats is saving in character. Renal retention of urea increases nearly from the onset of gestation and is more intensive in the goats carrying two foetuses, although the differences we found were not statistically significant. Despite the elevated tubular reabsorption of urea in both groups, its blood plasma concentration does not increase; even a falling trend can be seen over the last 3 gestational months (especially in twin pregnancy). This is most probably a result of an increased urea recycling in the lumen of the alimentary tract and an enhanced circulation of this metabolite within the blood-rumen-blood system.

Key words: kidney, single and twin pregnancy, excretion, urea.

INTRODUCTION

The final products of nitrogen metabolism are removed from a mammalian organism mainly by renal excretion. Nitrogen management in ruminants is different from that in humans or other non-ruminant animals. Urea recycling and its passage from the blood to proventriculi, which is referred to as rumen-blood-rumen nitrogen recycling, is a specificity of ruminant physiology [4,5,9,10]. The proportions between kidney-excreted urea and that returning to proventriculi vary and depend on the nitrogen balance of the system [2,20]. If high-protein feeds are provided, urea clearance is similar to that of inulin [10,26]. On the other hand, if low-protein feeds are fed, or during a period of increased requirement for proteins, urea clearance decreases and can represent as low as 10% of inulin clearance [10,26]. The processes of tubular reabsorption intensify to reach up to 87-95% of urea filtered load in some breeds of goats [26]. Pregnancy is a period of higher protein demand. Blood plasma urea and its excretion with urine in women decreases during pregnancy [14,15,19]. The reduction in plasma urea results from an increased glomerular filtration rate as well as from changes in the metabolism of the nitrogen compounds, to name just two factors among others. Hepatic urea synthesis decreases in pregnancy. According to Kalhan et al. [15], it is a result of a reduced supply of urogenous substrates to the liver and/or reduced activity of the ornithine-cycle enzymes [13,14]. Reduced synthesis of the basic urogenous amino-acids, i.e. alanine and glutamine, results from weakening of the transamination processes of branched-chain amino-acids, including leucine and isoleucine. During pregnancy, also the supply of α-amine nitrogen to the liver and transformations of amino-acids in the process of gluconeogenesis decreases [14,18]. Reduced plasma urea concentrations and lower urinal urea excretion have been observed also in pregnant sheep, cows, and goats [19,23,26]. Unlike in women, however, reduced plasma urea levels in pregnant goats result from enhanced urea circulation in proventriculi rather than from changes in glomerular filtration [18,26].

Studies on renal regulation of the nitrogen balance in pregnancy have been carried out mainly in women and laboratory animals. There are few reports on this issue in the available literature in relation to livestock animals. Renal function and the adaptive changes in the kidneys of pregnant goats have not been fully understood and still require much attention. Therefore, the presented studies have been undertaken in order to describe and compare changes in blood plasma urea concentration and urea renal excretion in single- and twin-pregnant goats, as well as to determine the mechanisms responsible for urea retention.

MATERIAL AND METHODS

The experiment was carried out on 16 pregnant White Improved goats (8 in single and 8 in twin gestation), aged 2–4 years. The animals did not show any clinical symptoms of a disease during the experiment. The goats were housed in individuals pens, fed 3 times a day according to the feeding standards [13], and were allowed to consume hay, straw, and water ad libitum.

The goats were mated in a natural way during the second heat (September/October), and the kiddings took place in the usual time (February/March). In about one month prior to the planned matings, as well as during the 3 first months of gestation, blood and urine samplings were carried out fortnightly; during the remaining two months, the material was collected weekly, always on the same weekday.

The animals were weighed before feeding, and catheterized into the external jugular vein about 9.00 a.m., which allowed swift and stressless collection of 5 blood samples in short, 20-minute intervals; the catheter was also used for administration of a precise dose of inulin, the test substance [27]. In order to precisely measure the diuresis volume (V), we placed Foley catheters inside the urinary bladders of the goats. Following a collection of the "zero" blood and urine samples (B0 and U0), the animals were intravenously administered 20 ml of 10-% inulin solution (2 g inulin) at 10.00 a.m. After another 10 minutes, urine collections started (U1, U2, U3, U4) taken in 20-minute intervals; in the middle of each interval a blood sample was also drawn (B1, B2, B3, B4). The blood was centrifuged immediately on the collection, and the resulting plasma, as well as urine samples, were frozen and stored at -20ºC until analysed. The blood plasma and urine were used to measure the concentration of urea by the colorimetry (Aqua-Medica laboratory biotests).

The following renal function parameters were calculated using the collected data: urea clearance (C), filtered load (F), tubular reabsorption (TR), and urinal excretion (UV). Calculated absolute values of these parameters were next converted to 1 m² of a goat's body surface area. We used the formula according to Meeh (after Ketz, 1974):

where:
BSA – body surface area (m²); BW – body weight (kg).

The detailed results were grouped by the type and month of gestation. Mean values (m) and standard deviations (SD) were calculated for the data. In order to test the significance of differences between the resulting data on renal function in each gestation period, as well as between the values of the same periods in single- and twin gestations, the data were processed statistically using one-way Anova with the multiple-range test (Statistica 6.0 package).

RESULTS

The means and significances of differences for the data resulting from the experiment have been presented in Table 1 . Blood plasma urea concentration in single-pregnant goats (Table 1) was relatively stable and ranged within 9.16-11.74 mmol·l^-1. In the other group of goats, we observe a decrease in urea levels starting from week 8 of gestation. The values observed at week 20 were significantly (P ≤ 0.01) lower as compared with those recorded during the 2nd month of pregnancy. In the last week of gestation, urea concentrations increased in this group, however, the change could not be statistically verified.

Table 1. Urea concentration in blood plasma (P) and clearance (C) in single and twin pregnant goats

week of pregnancy		P (mmol·l-1)		C (ml·min-1·m-2)
		single pregnant	twin pregnant	single pregnant	twin pregnant
0 (A)	SD	10.73 0.99	10.73 0.99	15.363G 7.871	15.363F.G 7.871
4 (B)	SD	11.19 1.31	11.78 0.70	14.033 4.767	12.711 5.964
8 (C)	SD	11.75 3.34	11.85F 1.94	13.367 4.969	12.729 5.172
12 (D)	SD	11.12 2.98	10.25 2.40	11.926 4.229	12.682F.G 6.681
16 (E)	SD	10.74 2.65	9.01 2.19	13.003G 4.891	12.642F.G 6.202
20 (F)	SD	10.24 2.88	8.43C 2.18	10.225 4.294	7.784A.D.E 2.949
21 (G)	SD	9.16 2.69	11.41 2.37	7.112A.E 2.563	6.399A.D.E 2.491

– mean SD – standard deviation A, C, D, E, F, G – significance of diferences (P≤0,01) of means particular weeks of pregnancy

Within the group of single-pregnant goats, renal urea clearance gradually decreased throughout the studied period, except for month 4 of gestation (Table 1). During the pre-gestational period, urea clearance was 15.363 ml·min^-1·m^-2, while in the last week before delivery it was as low as 7.112 ml·min^-1·m^-2 – the difference statistically significant (P ≤ 0.01). Similar changes in urea clearance were observed also in the twin-pregnant goats, although a significant decrease in this parameter in relation to the period before pregnancy occurred much earlier, with the beginning of the 5 month of gestation. During this month, as well as during the week prior to delivery, urea clearance was, respectively, 7.784 and 6.399 ml·min^-1·m^-2 and was nearly twice lower compared to that observed in non-pregnant goats, 15.363 ml·min^-1·m^-2. As mentioned before, the observed urea clearance variations had a similar pattern in both groups, thus no significant differences have been found between the groups during the same time periods.

The mean load of urea filtered to the primary urine in the single-gestation goats ranged between 0.461–0.688 mmol·min^-1·m^-2 (Table 2).  The parameter observed in the last week before birth were significantly (P ≤ 0.05) lower than those recorded at week 8 of gestation. In the twin-pregnant goats, urea filtered load remained within the range 0.425–0.691 mmol·min^-1·m^-2. Its level observed at weeks 16 and 20 were significantly lower compared with those at week 4. Glomerular filtration of urea in goats at week 20 of twin gestation was significantly (P ≤ 0.01) lower as compared with that observed in the other group during the same time.

Tubular reabsorption of urea  showed a distinct and statistically significant increasing trend in both groups over the entire period of the experiment (Table 2). The parameters observed at weeks 20 and 21 of single- (80.08% and 87.17%) and twin gestation (86.28% and 87.85%) were significantly (P ≤ 0.01) higher compared with those in non-pregnant animals (69.33%). Significant differences in urea tubular reabsorption were also found between week 21 and weeks 4 and 16 of single pregnancy, as well as between weeks 4 and 20 of twin gestation.

Table 2. Glomerular filtration (F), tubular reabsorption (TR), and excretion urea with urine (UV) in single and twin pregnant goats

week of pregnancy		F (mmol·min-1·m-2)		TR (%)		UV (mmol·min-1·m-2)
		single pregnant	twin pregnant	single pregnant	twin pregnant	single pregnant	twin pregnant
0 (A)	SD	0.590 0.085	0.590 0.085	69.33F.G 14.55	69.33F.G 14.55	0.1652G 0.0787	0.1652e.F.g 0.0787
4 (B)	SD	0.610 0.049	0.691E.F 0.107	74.59g 8.67	76.10 f 12.25	0.1556G 0.0486	0.1463E 0.0670
8 (C)	SD	0.688g 0.171	0.644 0.101	75.41 10.00	78.76 11.52	0.1934D.E 0.1209	0.1347E 0.0776
12 (D)	SD	0.573 0.142	0.499 0.274	77.94 8.70	78.98 11.26	0.1221C 0.0463	0.0995 0.0563
16 (E)	SD	0.578 0.165	0.427 B 0.175	76.20g 8.99	76.71 11.03	0.1399G 0.0656	0.0882a 0.0476
20 (F)	SD	0.606** 0.319	0.425B 0.136	80.08A 10.07	86.28A.b 5.41	0.1170 ** 0.0948	0.0591A.B.C 0.0337
21 (G)	SD	0.461c 0.202	0.528 0.225	87.17A.b.e 4.51	87.85 A 4.07	0.0637A.B.C.E 0.0450	0.0615a 0.0326

– mean SD – standard deviation a,b,c, e, f, g – significance of diferences (P≤0,05) of means particular weeks of pregnancy; A, C, D, E, F, G – significance of diferences (P≤0,01) of means particular weeks of pregnancy; ** significance of diferences – (P≤0,01) of means particular weeks in single and twin pregnancy

Non-pregnant goats removed with urine on average 0.1652 mmol of urea per minute per 1m² of body surface area (Table 2). Following a temporary increase at week 8 of single gestation, urea excretion gradually decreased. In the last week before birth, the parameter was nearly twice lower (P ≤ 0.01) compared with that in non-pregnant goats. A distinct reduction in urinal urea excretion was also observed in goats during a twin pregnancy; this parameter dropped nearly by half from the 16th week of gestation. If we compare urea excretion between the groups, the does at single pregnancy had slightly higher levels over the entire gestation, with a highly significant difference recorded at week 20.

DISCUSSION

In women, plasma urea concentration gradually decreases as the pregnancy develops; it falls significantly lower than in the pre-gestational period from as early as the first trimester of gestation [6,14,15]. Kalhan [14] reports that urea concentration in the plasma of non-pregnant women is approx. 5.1 mmol·l^-1, whereas during the 1st, 2nd, and 3rd trimesters of gestation, the parameter drops to, respectively, 3.4, 3.3, and 3.2 mmol·l^-1. The author states that   not only is the decrease in urea concentration a result of enhanced glomerular filtration, but also results from a reduced level of its hepatic synthesis. As a result of increased progesterone and oestrogens concentrations, the activity of the urea-cycle enzymes decreases; moreover, amino-acid consumption as energy source, i.e. the "transformation" of amino-acids into glucose through gluconeogenesis, decreases as well. These changes are accompanied by a reduced renal urea excretion level, which, however, is not related to its increased tubular reabsorption [12,14,15,18,21]. Opinions differ as to the pattern of changes in plasma urea concentration in ruminants. El-Sherif and Assad [7] state that plasma urea in pregnant ewes increases from week 10 of gestation. Similar observations in pregnant sheep have been reported by SAHLU et al. [24] and Brun-Bellut [2]. Most authors, however, state that in pregnant cows [19], sheep [3,8,23], and goats [11,22,26], plasma urea concentration and its renal excretion decrease. Freetley and Ferrel [8] have observed that urea concentration in pregnant ewes decreased from 5.59 to 3.83 mmol·l^-1 between weeks 1 and 20 of gestation, while Brzostowski et al. [3] report that the parameter in Polish Merino sheep was 9.38 mmol·l^-1in early gestation (first 2 months) and as little as 3.10 mmol·l^-1during the remaining period (the 4th and 5th months of gestation). A much smaller yet significant reduction in plasma urea concentration in pregnant goats, from 11.37 mmol·l^-1, in the 1st month, to 8.70 mmol·l^-1,  in the 5the month, was reported by Jankowiak et al. [11].

In both studied groups of goats, renal clearance and excretion of urea decreased gradually with gestation (Table 1, 2). These changes have resulted from a considerable gain in urea tubular reabsorption (Table 2). From the 1st month until week 21 of gestation, clearance and renal excretion of urea in both single- and twin-pregnant does decreased more than twice, with a slightly higher urea saving rate observed in the latter group. Despite a lower urinal urea excretion and a considerable reabsorption increase, blood plasma urea concentration did not rise; on the contrary, it exhibited a decreasing trend nearly until the end of gestation. This implies substantial recycling of urea in proventriculi, especially during the second half of gestation, where the foetus develops and gains in weight most intensely [2,26]. Increased renal urea retention enhances its circulation between the blood and the rumen. Pregnancy in goats and sheep is accompanied by a strong increase in urea tubular reabsorption and, in consequence, enhanced recycling of urea to the lumen of the alimentary tract [1,26].The fact that twin-pregnant goats had a lower urea concentration at weeks 16 and 20, as compared to the other group, confirms that the does had an increased requirement for nitrogen (protein) during the latter half of gestation. In order to retain urea in the system, ruminants have developed two mechanisms, i.e. reduction in glomerular filtration and increase in tubular reabsorption. The latter mechanism is used much more often and is of higher importance [2,26], which has been fully confirmed by the presented study.

The non-significant increase in plasma urea concentration observed in the last week of twin pregnancy is difficult to explain; it can be, however, probably attributed to energy deficiency occurring during this period due to a lower feed intake [16]. This enhances protein catabolism and its consumption as a source of energy. Deamination of amino-acids provokes additional urea synthesis.

CONCLUSIONS

To summarise the experiment, we have found that nitrogen management in the organism of a pregnant goat is economical in character. Renal retention of urea increases nearly from the very beginning of gestation and is more intensive in twin-pregnant goats, although the differences found have not been statistically significant. Despite an increased urea tubular reabsorption in both groups, plasma concentration of this compound does not rise or even decreases in the last 3 months of gestation, especially is twin pregnancy is the case. This most probably results from an increased urea recycling in the lumen of the alimentary tract and an enhanced circulation of the metabolite in the system blood-rumen-blood.

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Accepted for print: 4.03.2009

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Katarzyna Michałek
Departament of Animal Physiology and Cytobiology,
Faculty of Biotechnology and Animal Husbandry,
West Pomeranian University of Technology, Szczecin, Poland
Doktora Judyma 6, 71–466 Szczecin, Poland

Małgorzata Ożgo
Departament of Animal Physiology and Cytobiology,
Faculty of Biotechnology and Animal Husbandry,
West Pomeranian University of Technology, Szczecin, Poland
Doktora Judyma 6, 71–-466 Szczecin, Poland

Wiesław F. Skrzypczak
Departament of Animal Physiology and Cytobiology,
Faculty of Biotechnology and Animal Husbandry,
West Pomeranian University of Technology, Szczecin, Poland
Doktora Judyma 6, 71–466 Szczecin, Poland

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