Considering the BOHB and glycaemia values, at the moment of removing the sheep from fasting and the fact that there was no evidence of any clinical signs, it is reasonable to assume that these animals were affected by sub-clinical pregnancy toxaemia before treatments started [18, 19].
Early diagnosis is very important. The simplest method is the use of test strips. While it is important to bear in mind that the test strips used for the pregnancy toxaemia diagnosis is based on the semi-quantitatively determination of ketone bodies in urine, nevertheless these stripes react in presence of acetoacetate and acetone and do not detect BOHB which is found in larger quantity in this disease. The positive reaction of these test stripes clearly indicates the presence of ketonuria. However, there have recently appeared on the market portable and electronic devices (FreeStyle®, Precision Xceed®, Precision Xtra®, etc.) with great field level diagnostic potential, specially for veterinary clinicians, measuring blood BOHB in sheep [20], goats [21] or cattle [22]. There has been found a high correlation between the BOHB and glucose values measured with these devices and the ones obtained by the laboratory, showing high sensitivity and specificity [20, 22].
Taking into account that one of the main objectives of the treatment for pregnancy toxaemia is the increase of glycaemia [12, 14, 15, 23], when assessing the results in this paper we observed that once treatment started glycaemia began increasing in the three experimental groups. However, in sheep treated with propylene glycol and the ones treated with an i.v. infusion of glucose plus insulin, this increase took place earlier than in sheep treated with corn supplementation. This difference can be explained if we consider the different rates of absorption and conversion of the precursors used into glucose for treatment in groups A and B. According to Herdt and Emery [23] an i.v. infusion of glucose immediately causes an increase in blood glucose concentration resulting in transient hyperglycaemia, which we did not observe in our research as a consequence of the insulin subcutaneous infusion immediately after the glucose infusion, since this promotes the rapid glucose uptake and utilization by peripheral tissues [14].
Glycaemia increase in response to propylene glycol after 12 h of treatment from 1.79 ± 0.75 to 3.26 ± 0.48 mmol/L. Propylene glycol, which is mainly absorbed intact directly from the rumen at a rate of 40 % per hour [23] and reaches its maximum blood level within 30 min of administration and maximum blood glucose conversion at about 4 h after administration. Propylene glycol transformation in glucose probably occurs via conversion to pyruvate [23].
In this group, blood glucose values were the highest throughout the treatment. Propylene glycol produces a rapid increase of glucose, while glycerol is slowly degraded in the rumen producing a high proportion of propionate, main precursor of glucose via gluconeogenesis, resulting in a glycaemia increase for a relatively long period and furthermore, glycerol and propylene glycol treatment was repeated twice daily according to Rook [5], Wierda et al. [24] and Sienra et al. [25], all of which can explain glucose behaviour
In the group where fed corn (group C), glycaemia increased from 1.83 ± 0.64 to 2.00 ± 0.89 mmol/L, after 12 h of the start of the treatment, reaching 3.00 ± 0.72 mmol/L 48 h later. Ruminal microorganisms must attack the corn and transform it into volatile fatty acids, especially propionic acid [26, 27], which once formed, has to be absorbed at ruminal papillae level, being partly transformed in lactate in the rumen wall and both are converted into glucose in the liver via neoglucogenesis. Between 18 and 42 % starch from corn may escape rumen degradation and be digested in the small intestine [28]. However, only 30 to 35 % of the glucose formed from starch intestinal digestion, can be found in the portal vein [29].
The treatment of the sheep with intravenous glucose did not reach stable glycaemia concentrations, rapid increases post-administration were produced, followed by important decreases thereof. González-Montaña et al. [30] and González-Montaña et al. [31] described this glycaemia behaviour and attributed it to glucose renal excretion when it was administered intravenously. Intravenous infusions of glucose solutions is bound to result in transient hyperglycemia leading to diuresis and urinary loss of a large portion of the administered glucose [23]. Fox [32] established that after an i.v. infusion of glucose, approximately an 80 % of the dose is excreted by urine.
Considering that another of the objectives of pregnancy toxaemia treatment is to restore blood ketone bodies to normal concentrations [12, 23], results in this research showed that while blood BOHB decreased in the three experimental groups, once treatments were implemented, that decrease was sharper and earlier than in sheep treated with propylene glycol, and in addition BOHB remained significantly lower throughout the research. We think that the fatty acids metabolism was altered suppressing its mobilization from the adipose tissue, reducing their entry into the liver and reducing their transformation into mitochondrias. Propylene glycol increases pyruvate concentration with the subsequent oxaloacetate production via pyruvate carboxylase. Available oxaloacetate increase is expected to produce an increase in intramitochondrial citrate concentration. Intramitochondrial citrate escapes to form malonyl-CoA when is increased, it is a powerful transformation suppressor of fatty acids into mitochondria [33–35]. The fatty acids entry reduction into hepatic mitochondrias results in hepatic ketosis decreasing. Additionally, glucose supply provided by propylene glycol treatment may increase the insulin:glucagon relationship therefore affecting ketosis [23].
Glucose infusion results in a gluconeogenesis reduction [23], leading to an increase of the Krebs cycle which intermediates concentration inside the mitochondria due to the smaller quantity of substrates that are transported to the cytosol serving as glucose precursors. Citrate is one of those intermediaries, when increases it also increases Malonyl-CoA formation [33–35]. Herdt and Emery [23] also add that a glucose injection may cause a reduction in glucagon plasma since its secretion decreases in response to hyperglycaemia.
Increased citrate (and malonyl-CoA) and glucagon reduction will cause the fatty acid entry reduction inside the hepatic mitochondria. To these effects in the ketone bodies synthesis reduction we must add the antiketogenic effects of insulin, which was administered together with the intravenous glucose. The antiketogenic effect of insulin causes the following actions to occur: 1) it depresses the release of fatty acids from adipose tissue; 2) it promotes the use of ketone bodies in peripheral tissues; 3) it suppresses the entry of fatty acids to the liver; 4) relatively low insulin concentrations block the ketogenic effect of glucagon, thus limiting the fatty acids entry into the mitochondria [23, 36].
While the three treatments applied were able to restore blood glucose and BOHB normal concentrations, glycerol + propylene glycol treatment was the one, which achieved the results in less time and it had a more prolonged effect. Propylene glycol treatment results administered in the same doses and frequencies as in this research, in animals with clinical pregnancy toxaemia showed contradictory results. While Sienra et al. [25] indicated good results and metabolic parameters normalization, Wierda et al. [24] only achieved good results in mild forms of the disease, reporting that in animals with advanced pregnancy toxaemia results were poor. Sargison et al. [1] and Brozos et al. [14] suggested that although given a complete treatment, only one third of sheep with clinical pregnancy toxaemia would probably survive Koenig and Contreras [37] reported that in sheep induced to toxaemia by fasting, propylene glycol treatment reduced mortality in about 50 %. The differences found are explained by considering Rook’s proposals [5]. This author suggested that precursors compounds of glucose synthesis in the liver would be useful provided there is no organ severely compromised. Herdt and Emery [23] added that the ability to use propylene glycol is reduced in liver fatty infiltration. According to our findings and to Marteniuk and Herdt [15], the treatment with propylene glycol should be initiated as soon as possible, before as complications of the disease appear (acidosis, irreversible neurological damage, severe dehydration, kidney failure, etc.) and the chances of recovery would be lower.
Regarding corn treatment we can make the same suggestion considering the results in this research. Marteniuk and Herdt [15] stated that sheep in early stages of the disease and still maintaining their appetite, the energy increase supplied by the starch in the diet would be enough to reverse the conditions, adding that a more vigorous treatment is needed when animals are not eating or are eating very little. This type of treatment would be of practical use in subclinical pregnancy toxaemia cases in a commercial flock.
Koenig and Contreras [37], Cal Pereyra et al. [10] and Lima et al. [7] noted that advanced cases of clinical pregnancy toxaemia caused hyperglycaemia, thus glucose based therapy would have no effect at that stage of pregnancy. In spite of this fact, our findings show that at the early stages of pregnancy, treatment with intravenous glucose and subcutaneous insulin in pregnancy toxaemia is useful to reverse the process. However, according to Rook, [5] in considering the usage of this treatment, it would be of little practical use in cases of pregnancy toxaemia in conventional flocks but it would be justified in individual cases of the disease, such as in hospitalized and highly reproductive valued animals.
