The nutrition partition during the periparturient period put considerable strain on animal health. This strain is considerably increased by intensive farming methods adopted for higher quality products increasing the susceptibility to peripartum disorders. In the present study glucose concentrations were higher in lactation than pregnancy and similar findings were reported by other researchers (Balikici et al. 2007; Moghaddam and Hassanpour, 2008; Taghipour et al. 2011). Increased glucose levels after lambing indicate that ewes feed intake has recovered and her energy status has improved. Glucose is the main source of energy for the developing fetus as well as for placenta, uterine tissue and supporting membrane which together put heavy demand on maternal glucose supply during late pregnancy (Khan and Lundri, 2002; Magistrelli and Rosi, 2014). The last six weeks of gestation accounts for mora than 50% of fetal growth (Mohammadi et al. 2016), and during this period fetal glucose metabolism account for 40–70% total glucose metabolized in sheep, resulting in low systemic glucose concentration as observed in late part of gestation.
After lambing plasma glucose levels were higher in PP ewes compared to MP. The lactating mammary gland uses the major part of circulating glucose for lactose production. Due to incomplete mammary gland development in primiparous animals, the mammary glucose uptake is low, resulting in greater systemic glucose level (Magistrelli and Rosi, 2014).
Despite the fact that glucose is the principal metabolic fuel and is required for crucial organ function, particularly foetal growth and milk production, it remains an insensitive indicator of energy status due to its tight homeostatic regulation (Rayan et al., 2019). Monitoring the energy status of pregnant sheep by measuring serum NEFA concentration is an alternative and useful technique.
During the later pregnancy as the required increase in glucose production to fulfill the demands of developing fetus and extrauterine tissue may be insufficient, mobilization of the lipid reservoir and increase free fatty acid content in the blood occurs to meet the energy demands. The significant increase in plasma NEFA concentration during last month of gestation and early lactation indicates that the animals were in negative energy balance (NEB). To overcome this NEB the body mobilize the fat to compensate for the shortage in energy needed, resulting in an increase of NEFA concentration in blood (Caldeira et al. 2007). The increase in NEFA in the late pregnancy and early lactation coincided with the decline in glucose concentration, and this type of adjustment is necessary to meet the energy demand of growing fetus and mammary gland for lactogenesis and increased milk secretion (Samira et al. 2016). The rise in NEFA levels coincided with a drop in glucose levels in late pregnancy and early lactation, and this adjustment in metabolism is required to fulfil the energy demands of the growing foetus and mammary gland for lactogenesis (Samira et al., 2016).
Comparing the two groups, NEFA was significantly high one week pre-lambing in PP while MP ewes had significantly high NEFA at 1-week post-lambing. Animals in early parties are still in the growing stage and require nutrients both for the growth of fetus and the animals itself (Wathes et al. 2007), which leads to a significant decrease in glucose and increase in NEFA in last stage of pregnancy as observed in PP ewes in the present study. Thus, the increased need of energy in early parities causes increased mobilization of body fats leading to the increased NEFA concentration. With increasing parities, udder development naturally increases, resulting in steadily increased milk production and around the fourth or fifth lactation, the maximal milk yield is reached (Pavlicek et al. 2006; León et al. 2012; Magistrelli and Rosi, 2014; Abraham et al. 2017). Thus, the significantly high NEFA concentration and corresponding low glucose concentration observed in MP in post lambing could be attributed to the more energy demand for milk production in MP ewes and hence more mobilization of body reserves for synthesis and maintenance of milk during early lactation.
A significant change was observed in the TPP and albumin levels, with concentration reaching its lowest value one-week post-lambing. Brozostowski et al. (1995) also reported decreasing trend of TPP and albumin levels during late pregnancy and gradual increases towards the end of lactation. Since ruminants' hepatic gluconeogenesis is predominantly accomplished using gluconeogenic amino acids, the reduction in TPP and albumin levels with the progression of pregnancy may be attributed to the greater protein and energy requirements for gestation (Balikci et al. 2007). Protein is the primary nutrient for uterine tissue during the last stages of pregnancy (Schmitt et al.2018), and protein synthesized by fetus are made from amino acids obtained from dam (Antunovic et al.2002; Schmitt et al.2018) and during this period, the foetus tissues, particularly muscle, grow exponentially, resulting in a corresponding decrease in maternal protein levels. The immediate decrease in TPP after lambing could be attributed to the removal of γ-globulin from the blood for milk secretion after parturition (Cepeda-Palacios et al. 2018). Celi et al. (2008) reported that total proteins are significantly low after parturition and contributed to the removal of γ-globulin from the maternal circulation.
During late stage of gestation, numerically low TTP and albumin levels were observed in PP ewes had, while as reverse was observed in MP which had numerically low TPP and albumin during lactation and this could be due to the fact that PP ewes need nutrients for their growth as well as the growth of fetus resulting into more drain of available proteins in blood (Wathes et al. 2007). Morever, albumin is mostly directed toward foetal development tissue, whereas globulin is utilized in the production of milk (Balıkcı et al. 2007; Karapehlivan et al. 2007; Obidike et al. 2009), resulting into more decrease of albumin pre-lambing in PP compared to MP.
Urea and creatinine are constituents of nitrogen metabolism (Cepeda-Palacios et al. 2018), and their increased levels are associated with kidney damage; however, their decreased concentration is related protein and energy levels in diet (Samira et al. 2016). Plasma urea level is a significant indicator of dietary protein intake, synthesis, and degradation in both sheep and goats (Schroder et al. 2003). In the present study significant decrease in urea concentration was observed from 4 weeks prelambing to 1 week post-lambing when urea levels were lowest, however no significant change in creatinine was observed except at one week post-lambing when level were highest. The decrease in BUN could be due to increased urea recycling into the digestive tract or better nutrition management (Gürgöze et al. 2009) or the use of urea for protein synthesis on the rumino-hepatic route, as reported by Yokus and Cakir 2006 in cattle, to compensate for inadequate protein uptake during late gestation. The amount of creatinine secreted daily remains unaffected by diet, age, sex or exercise but is a function of the muscle mass (Njidda et al. 2013). High need for energy by ewe during lactation leads to an increase in protein catabolism which increases blood creatinine level to an extent above the ability of kidneys to eliminate (El-Sherif and Assad, 2001) and thus the observed increase in creatinine one-week post-lambing might have been because of high protein catabolism during this stage as corresponding protein levels were lowest one-week post-lambing.
During the periparturient period, liver and kidneys are in hyper-function (El-Sherif and Assad, 2001), resulting in the corresponding biochemical changes in the blood. In the present study, there was a significant change in AST and GGT level. The activity increased steadily towards the end of gestation and reached the highest activity in the immediate post-lambing period. The activity of AST provides an estimate of liver function (Donia et al. 2014) and is best associated with impaired hepatic function in fatty liver disease and has been used in herd monitoring programmes to detect fatty liver disease. Reduced dry matter intake and consequent increase in fat mobilisation during the peripartum period, leading to hepatic lipidosis and hence affecting liver function, could be the cause of altered enzyme activity(Greenfield et al. 2000). To provide the energy and protein requirements for the onset and maintenance of milk synthesis, there is an intense burden on the liver (Roubies et al. 2006) in lactating ewes, resulting in increased liver enzymes as observed in the present study.
GGT is a membrane-bound enzyme found in cells with higher rates of absorptive or secretory capacity. Although GGT activity is seen in many organs, it is predominantly used as a serum marker in animals to diagnose liver illness (Milinković-Tur et al. 2005). The mammary gland's GGT activity is also significant and during milk synthesis initiation and maintenance, GGT is released from the alveolar cell membrane into colostrum or milk, varying its activity in serum (Ramos et al. 1994). A small part can reach the blood, which will contribute to the increase in serum level However, the major part comes from the liver because of its over activity during the peri-parturient period. Since initiation and maintenance of milk production are directly related to GGT levels, the probable significant increase in MP at1 and 4 weeks post-lambing could be because of the increase in milk production in MP ewes.
Besides liver specific enzymes, plasma bilirubin is also an indicator of liver injury (Lubojacka et al. 2005). The present study revealed a significant increase in bilirubin concentration in late gestation and early lactation. A similar finding was reported by Bertoni and Trevisi, 2013, who observed a significant increase in bilirubin concentration during the peri-parturient period in dairy cows. Bilirubin is not a protein, but its clearance is due to some liver-specific enzymes. Its increase is probably because of the lower synthesis of enzymes responsible for its clearance, which mainly occur during the liver insult.
During pregnancy, serum cholesterol and triglyceride levels gradually declined and reached their lowest levels after lambing. Cholesterol is synthesized in the small intestine epithelium for the transportation of dietary lipids; therefore lower plasma levels may be expected because of lower dry matter intake around the periparturient period (Douglas et al. 2006). Also, cholesterol is the precursor of various steroid hormones whose concentration increases in late gestation (McDonald et al. 2002). During late gestation, utilization of cholesterol by fetus (Guédon et al. 1999) increases, resulting in less plasma cholesterol levels. Cholesterol is also an important component of milk, and during lactation, an increase in nor-epinephrine and epinephrine production stimulates free fatty acid mobilisation, whereas lipogenesis and esterification are inhibited, resulting in a drop in cholesterol levels in the immediate post-lambing period. (Nazifi et al. 2002; Tanvi et al. 2016). HDL constitutes about 60% cholesterol (Sevinc et al. 2003), so the observed HDL change in the present study could be due to a corresponding decrease in cholesterol levels.
A significant decrease in serum triglycerides was observed one week pre- and post-lambing. This drop could be interpreted as the result of increased lipolysis, which is regulated by hormones, and not an indication of energy insufficiency. The NEFA extracted by the liver are oxidized or esterified into triglycerides, and either exported in very low-density lipoproteins (VLDL) or accumulated in liver tissue, and ruminants have lesser capability to synthesize and secrete VLDL from the liver, but a similar capacity to reconvert NEFA back to TGs (Graulet et al. 1998). Thus, the imbalance of the liver’s ability to uptake fatty acid and its capacity to secrete lipoproteins synthesized from triglycerides (Pysera and Opalka, 2000) decreases triglyceride levels. Moreover, the circulating triglycerides also contribute considerably to synthesis of milk fat (Nazifi et al. 2002; Tanvi et al. 2016). Thus, the observed decrease one-week pre and post lambing could also be due to the mobilization of triglycerides for initiation and maintenance of milk synthesis during early lactation.
Plasma Ca and P levels were significantly decreased in the last month of gestation and continued to decrease up to one week post lambing. Calcium levels required for pregnancy and lactation are much higher than those for maintenance, therefore to meet the increased requirements at tissue level, Ca and Pi absorption from the gastrointestinal tract and resorption from bones should increase (Donia et al. 2014). However, during high demand of pregnancy and lactation, this process is unable to balance the loss of ions from blood, and hence concentrations of these ions decrease (Elnageeb and Adelatif, 2010). Thus, the increased requirement of calcium for fetal skeleton mineralization during late gestation and increased secretion of Ca in milk during early lactation (Liesegang et al. 2007; Antunović et al. 2017) coupled with less dry matter intake results in decreased calcium concentration. The decreased phosphorus has been attributed to a decrease in dry matter intake and increased utilization to enhance carbohydrate metabolism of pregnancy. Moreover, it has been reported that with the increase in milk production, more phosphorus from the ingested amount is transferred to milk and less is secreted with faeces, causing more drop in blood phosphorus levels (Valk et al. 2002) and this might have resulted in low phosphorus concentration in immediate post-lambing in the present study. Parity was found to affect minerals levels, PP ewes had low Ca and Pi pre-lambing than MP, while MP had less mineral post-lambing. The Ca and Pi requirement is more in young ones for skeletal growth and since the PP animals besides having increased demand of minerals for mineralization of fetal skeleton are themselves in their active growing stage resulting in more drain of mineral during in them (Wathes et al. 2007).
Though there was no significant change in sodium levels with the time, potassium levels decreased significantly in the last month of gestation. Elnageeb and Adelatif (2010) reported that potassium levels decreased significantly during late gestation and attributed these changes to decreased plasma progesterone and increased aldosterone levels, resulting in more potassium excretion hence decreased levels in the blood.
Plasma Fe levels decreased during late pregnancy and continued to fall 1 and 4 weeks post-lambing. The drop in plasma Fe levels observed during late pregnancy and early lactation may be due to the fetus's high need for iron. Similar findings were reported by Yokus and Cakir (2006) and Tanritanir et al. (2009). In blood, iron is mainly bound with proteins called transferrin and ferritin, and the amount of ferritin in maternal blood has been considered to indicate the amount of Fe stored in the body, and its concentration falls as pregnancy advances. During pregnancy, substantial quantity of ferritin is deposited on placental villous tissue and gets integrated into the placenta via pinocytosis in the trophoplast, thereby lowering its maternal blood levels (Swenson and Reece(1993).
Physiological status affects the zinc levels, with decreased zinc along the gestation and lactation periods (Elnageeb et al. 2010). Developing feotus accumulates almost 1 to 2 mg of Zn/ day. The demand for zinc in later gestation increased many fold when feotus is growing exponentially (Donia et al. 2014; Elnageeb et al. 2010), resulting in the decreased concentration of zinc in maternal blood. Zinc is primarily bound to albumin and the change in albumin concentration may have a significant effect on Zn levels. In the present study, the albumin one-week post-lambing was lowest resulting in a corresponding decrease in the Zn concentration (Elnageeb et al. 2010). Moreover, there is also a heavy loss of Zn in colostrum and milk (Pavlata et al. 2004) which might have led to a further decrease in Zn concentration in post lambing.
Similar to zinc, blood Cu status also fluctuates during the periparturient period. The increase in copper concentration during late pregnancy could be related to high progesterone levels or the fetal demands and mobilization of stored maternal copper for the development of the nervous system (Elnageeb et al. 2010). The immediate post-partum period is often stressful, and stressed animals' blood levels of Cu and ceruloplasmin, a Cu transport protein, are frequently high (Ward and Spears 1999). Ceruloplasmin is an acute phase protein and its levels rise in response to injury, infection, and inflammation. This could explain why the blood level of Cu was higher in post lambing, as lambing and immediate lambing is a stressful period with tissue damage, such as in the uterus. (Meglia et al. 2010).