​Mineral Analysis in the Amniotic Fluid of Gaddi Sheep during Different Stages of Gestation

Amniotic fluid, which is swallowed by the developing fetus, is an important source of certain minerals and trace elements available for fetal absorption and fetal growth (Jalali and Koski, 2018). These trace elements in amniotic fluid are components of numerous regulatory enzymes and hormones essential for division and differentiation of fetal cells (Lewicka et al., 2017). The concentration of various minerals and elements in amniotic fluid change over the course of pregnancy, with gestational age and fetus growth (Suliburska et al., 2016). Gaddi sheep is an important livestock breed of northern temperate region of India. Limited study has been done on the foetal fluid of Gaddi sheep (Shukla et al., 2019; Shukla and Rajput, 2020). The aim of the present study was to evaluate the concentration of some micro and macro minerals in the amniotic fluid and their relation to fetal age and fetal weight.

This study was conducted at Department of Veterinary Anatomy, Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishwavidyalay, Palampur from 2015 to 2019. Fifty normal gravid genetalia were collected from local slaughter houses located in and around Palampur (Himachal Pradesh) from apparently healthy pregnant Gaddi sheep. 10 ml of amniotic fluid was collected in labelled plastic tubes after puncturing the amniotic sac. The collected samples were stored at -18°C. The foetal membranes were separated and each foetus was weighed in grams on analytical balance (Table 1). The approximate age was computed using the formula:
 
                          Y = 2.74 X + 30.15          (Gall et al., 1994) /> Where
Y= The developmental age of foetus in days.
X= The crown-rump length in cm.
 

       
The gestation period was divided into four groups according to age of foetus viz. group I (30-60 days), group II (61-90 days), group III (91-120 days) and group IV (120 days to term).
       
Different macro (magnesium and calcium) and micro (iron, copper and zinc) minerals were estimated by atomic absorption spectrophotometer and the values of sodium and potassium were estimated by flame photometry (Mediflame Systronics Flame Photometer-127). Data was statistically analysed by one way analysis of variance using SPSS ver. 17.0 (P<0.05).

In this study the mean concentration of sodium in the amniotic fluid of Gaddi sheep decreased non significantly (p<0.05) from group I to group III and then remained same in group IV of gestation (Table 2). Aidasani et al., (1993) stated that the mean concentration of sodium was 37.24±3.89 mEq/L in the amniotic fluid of goat. The significant negative correlation was observed between sodium concentration and foetal weight in group II (Table 4). However, non significant negative correlation was observed between the concentration of sodium and the age of foetii and between sodium concentration and foetal weight in group III vs IV and in group I and III (Table 3, 4), respectively.

 

 

       
The average value of potassium decreased significantly from group I to group III and increased in group IV of gestation similar to findings of Prestes et al., (2001) in sheep and Khadjeh et al., (2007) in goats. The mineralocorticoid activity due to intrauterine fetal maturity acted on fetal kidneys, which was believed to be responsible for increasing potassium and decreasing sodium concentrations in fetal urine by same researchers. The correlation coefficient was highly significant between the concentration of potassium and the age of foetii in group II vs III and negative non significant in group II vs IV and III vs IV. The correlation coefficient between potassium concentration and foetal weight was negative in group II and IV, although it was non significant (Table 4).
       
The mean concentration of chloride remained consistent in group I and II of gestation, increased slightly in group III and then decreased significantly in group IV of gestation. Similar observations were made by Prestes et al., (2001) in sheep and Khadjeh et al., (2007) in goat. The significant positive and negative correlation was observed between concentration of chloride and foetal weight in group I and group II of gestation respectively (Table 4). However, correlation observed between the concentration of chloride and the age of foetii was negative and non significant in groups I vs III, II vs III and III vs IV (Table 3).
       
Calcium concentration did not vary significantly among different groups of gestation. The correlation in the concentration of calcium and foetal age was highly significant in group I vs II and negatively non significant in groups I vs IV, II vs IV and III vs IV. Also negative non significant correlation was observed between calcium concentration and foetal weight in group II and III of gestation.
       
Phosphorus concentration did not vary significantly from group I to group III and then it increased significantly in group IV of gestation. Aidasani et al., (1993) reported that the average concentration of phosphorus in the amniotic fluid of goat was 4.31±1.31 mg/dl. The negative non significant correlation was observed between its concentration and foetal age throughout gestation (Table 3) and between its concentration and foetal weight in group II and group IV of gestation (Table 4).
       
The mean concentration of copper remained nearly consistent throughout gestation. In humans, fetal copper concentration reportedly increased or remained stable throughout gestation (Hefnawy et al., 2011) where as Parkinson et al., (1981) found that amniotic fluid copper concentration gradually increased during pregnancy. Copper is essential for development of CNS in embryos (Hefnawy et al., 2011). Copper is a component of superoxide dismutase, catalase and cytochrome oxidase and as such plays an important role in oxidation reactions. In addition, by contributing to the formation of ceruloplasmin, copper plays an important role in the absorption and me­tabolism of iron (Lewicka et al., 2017). The correlation of its concentration with foetal age and with foetal weight was negative in group II vs IV and groups I, II and IV respectively, although it was non-significant.
       
The mean concentration of iron decreased significantly in group III of gestation as compared to other groups. It participates in redox reactions, immune processes and DNA synthesis, as well as being a substrate in the synthesis of heme, the main component of hemoglobin (Lewicka et al., 2017). The correlation between the concentration of iron and age of fetii was highly significant between group I and group II (Table 3). Negative non significant correlation was observed between its concentration and fetal weight in group I and group III of gestation.
The mean concentration of zinc in the amniotic fluid of Gaddi sheep increased significantly in group IV of gestation as compared to other groups. Zinc has an important function in the body par­ticipating in RNA, DNA and proteins synthesis, as well as playing a role in the stabilization of cell membranes and the skeletal system (lewicka et al., 2017). The correlation of its concentration with fetal age was significant between group I and group III. Its concentration was negatively correlated with fetal weight in group II and III of gestation, although the correlation was non-significant. The availability of zinc in forage is not sufficient so its supplementation in forages and concentrates is important to meet the daily requirements of production and reproduction (Farah et al., 2013). Copper, zinc and iron are essential heavy metals and any imbalance in their interactions during pregnancy could have a major impact on fetal growth and development (Olfati et al., 2016).
       
Magnesium concentration in the amniotic fluid increased significantly from group I to group IV of gestation. Aidasani et al., (1993) found the concentration of magnesium to be 2.53±0.24 mEq/L in the amniotic fluid of goat. An optimal level of magnesium in serum is effective in lamb’s growth and health and thus prevents the occurrence of diseases related with hypomagnesemia (Olfati et al., 2016). Negative non significant correlation was observed between its concentration and foetal age in group III vs IV (Table 3) and between its concentration and foetal weight throughout gestation (Table 4).
               
The variation in the concentration of different minerals in amniotic fluid of Gaddi sheep with other studies might be due to several factors such as gestation age, species, breed, rearing and environmental conditions, nutritional status of the animal and the analytical methods use which can affect the composition of the amniotic fluid. Amniotic fluid usually reflects the distribution of the minerals in the body (dam/fetus) at the moment sample is taken. Their deficiency in dams results in insufficient maternal transfer of these minerals to fetus which can hamper the normal growth and development of fetus (Olfati et al., 2016). Therefore, amniotic fluid can provide important information to a clinician that may not be readily available with blood analysis.