Trace Elements in Pregnant and Non-pregnant Sheep and Goats and Their Relation to Steroid Hormones

Trace elements are essential intermediaries in ovarian function and their involvement in both follicular development and hormone production is well documented (Yaman et al., 2007) in man and animals. Trace elements are found in different ovarian structures including mature follicles and corpora lutea even atretic follicles (Bhardwaj and Sharma, 2011). Iron for instance is indispensable for the ovulation process (Chavarro et al., 2006). Trace elements are necessary for maturation of the oocyte and maintaining the number of stored follicles at their normal range through their inevitable role in gonadotropins secretion (Chavarro et al., 2006; Singer et al., 2011). As a key player in the biosynthesis of cholesterol, they are irreplaceable for steroidogenesis and subsequent regulation of the folliculogenesis in female animals (Amin et al., 2016). Accordingly, animals receive trace elements-deficient diets are prone to failure of oogenesis, increased follicular atresia, inadequate steroidogenesis, ovulation failure and CL persistence (Gürdoðan et al., 2006; Vázquez-Armijo et al., 2011; Grace and Knowels, 2012; Ceko et al., 2016). Their association with the changes in the different ovarian events and the accompanied fluctuation on the level of steroid hormones during the reproductive cycle is crucial for the area of research interested in in vivo and in vitro manipulation of the mammalian oocyte. In this respect, further investigations are needed to fulfill this concept.
       
The normal requirements of trace elements increase greatly during pregnancy and adequate supply is of utmost importance before intended breeding to prepare the animal for the sequences of pregnancy and associated physiological changes during this period. Trace elements are involved in the growth, development and normal evolution of the pregnant uterus and its contents (Amin et al., 2016). Their roles in the enzymatic activity, efficiency of the immune system, the buildup of antioxidant capacity, integrity of the inner lining of the reproductive organs are non-negotiable (Uslu et al., 2017). Abortion, retarded growth, pre-term birth, stillbirth and birth of congenitally-deformed newborn are the result of trace elements-deficient diets. There were mixed responses to reports on the association between pregnancy and trace elements, regardless of the fact that the requirement for trace elements doubles or triples during pregnancy (Nawito et al., 2015; Spencer et al., 2015).
       
The present study aimed to study the association between the level of four major trace elements known for their deficiency in Qassim region; manganese (Mn), selenium (Se), iron (Fe) and zinc (Zn), ovarian findings, stage of pregnancy and level of steroids in non-pregnant and pregnant sheep and goats, respectively.

Total of 129 ewes (68 pregnant and 61 non-pregnant) and 54 does (32 pregnant and 22 non-pregnant) in Qassim region, Saudi Arabia, were included in this study during the breeding season (autumn/winter, 2020). The study was carried out at the university teaching hospital, Qassim University. Animals aged 1-5 years, weighed 55-65 Kgs, average body condition score 3 (Kenyon et al., 2014) fed maintenance ration for breeding females (NRC, 1985 and 2007) and drinking water ad libitum.
       
The external genitalia of the studied animals were evaluated for their soundness. Transabdominal and transrectal ultrasonography using a 5 MHz transducer (Eickemeyer - Medizintechnik für Tierärzte KG, export office, Eltastraße 878532, Tuttlingen, Germany) for monitoring the reproductive tract of the studied ewes was carried out to diagnose pregnancy and to determine the ovarian findings in non-pregnant animals (because animals were not in the same stage of the cycle they were classified according to the ovarian findings). The built in software was used to determine the stage of pregnancy after freezing the screen at the best-fit sonogram for the placentomes or the fetal parts designed to determine the stage of pregnancy. The pregnant animals were categorized according to stage of pregnancy into 1 to 2, >2 to 3, >3 to 4 and > 4 months groups. Non-pregnant animals were, further, categorized according to the findings on their ovaries into those with corpus luteum (CL) vs. corpus luteum and large follicle [CL+F (>5 mm)] vs. growing follicles (GF, <5 mm) vs. mature follicle (MF, >5 mm) groups.
       
On admission, animals were venipunctured and bled into plain tubes at 8:00-10:00 a.m. and let at room temperature for 30 minutes. Centrifugation of the collected samples at 3000 xg was carried out for 15 minutes. The obtained serum was kept at -20°C until hormonal assay and estimation of trace elements.
       
Flame emission atomic absorption (Moffat et al., 1986) was used to estimate serum levels of Mn, Se, Fe and Zn after samples digested using HClO₄-HNO₃ mixture according to the technique described previously (Antoniou et al., 1995).
       
Levels of E2 and P4 in serum were determined by ELISA (kits brought from Prechek Bio., Inc., Young In city, Korea, Lot No: 119011502). The coefficients of variation of the intra-assays were 3.7 and 5.1%, inter-assays 6.4 and 6.8% for E2 and P4, respectively. Three pg/ml for E2 and 0.12 ng/ml for P4 were the sensitivity of the assays.
       
The data were expressed in means±SE. SPSS program, version 25 (SPSS Inc., Chicago, IL, USA, 2017) was used for statistical analysis. One way-ANOVA was used to test the difference between animals’ groups. Relationships between trace elements and steroids were tested using the correlation coefficient. The level of significance was set at P<0.05.

Trace elements were lower in pregnant compared with non-pregnant ewes and does. This decrease was significant for Mn (P=0.01), Fe (P=0.05) and Zn (P=0.001) in ewes (Table 1) and for Fe (P=0.03) in does (Table 2). Positive association between Fe and P4 (r=0.56; P=0.01), Fe and E2 (r=0.65; P=0.03) and Mn and P4 (r=0.61, P=0.04) in pregnant ewes. In the present study, Mn, Fe and Zn in sheep and Fe in does decreased significantly during pregnancy compared with open ewes and does. Though non-significant, Se decreased also in pregnant animals. These decreases were more obvious with the advancement of pregnancy. Similarly, it was found that the concentration of Se and other elements in the circulation falls considerably (12%) during normal pregnancy as gestation progresses in sheep (Ganie et al., 2014; Uslu et al., 2017). Another potential reason of this fall in serum trace elements in blood during pregnancy is these elements may be transported to the fetus by certain mediators, which are expressed in the placenta, thereby causing a fall in blood concentration of these elements during pregnancy (Makhlouf et al., 2020; Derar et al., 2022). It has been speculated that progesterone mobilizes trace elements towards the reproductive organs especially the uterus particularly during pregnancy (Dalai et al., 2017) which may explain the decline in serum trace elements during pregnancy and the CL-dominant compared with the follicular-dominant statuses in non-pregnant animals in the present study. In addition, the reduction in trace elements during pregnancy may be the consequence of hemodilution from maternal plasma expansion, increased transport of these elements to the fetus and as a result of increased utilization for the synthesis of elements-dependent antioxidant proteins required to combat the increased oxidative demands of pregnancy (Spencer et al., 2015). Both Fe and Zn are involved in the epithelialization of the fetus. Zn is essential for DNA replication and protein synthesis of the fetal tissue not mentioning its role in the immune mechanism during the gestation period (Nawito et al., 2015; Dalai et al., 2017; Makhlouf et al., 2020).
 

 

       
Ewes with MF in their ovaries had higher Se (P=0.02) and Fe (P=0.001) compared with ewes having other structures in their ovaries (Table 3). Fe (P=0.001) and Zn (P=0.000) were higher in does having MF compared with does having other structures either GF or CL (Table 4). A positive association was found between E2 and serum Fe in non-pregnant ewes (r=0.7, P=0.03) and does (r=0.54, P=0.05).
 

       

 
Presence of mature follicles in the ovaries of ewes was associated with higher level of serum Se and Fe in the present study. Similarly, healthy follicles of different size categories from small to large size were associated with a rise in serum concentration of these elements in goats (Dalai et al., 2017). It was postulated that trace elements are essential for ovarian tissue build-up involving mitochondrial function, cell division, biosynthesis of estrogen and progesterone and combating free radicals (Gajda et al., 2008). These elements exert their action via induction of DNA synthesis, activation of adenyl kinase, phosphodiesterase, membrane–bound adenyl cyclase and lipid peroxidase in the process of follicle growth and maturation (Zheng et al., 2015).
       
Females with delayed sexual maturity, idiopathic infertility were found to have inadequate Se and aberrated Se-binding protein-1 (Edassery et al., 2010). Fe, Se and Zn were consistently confined to the granulosa cell layer of different classes of the ovarian follicles including small, medium and large follicles (Ceko et al., 2015). It was reported that the elevated Se in healthy follicles is may be attributable to the upregulation of selenoproteins GPX1 in these follicles compared with atretic ones (Ceko et al., 2016). Based on these findings, there is strong evidence that these elements are decisive for different stages of follicular development including recruitment, selection, dominance and maturation (Edassery et al., 2010). It was found that Se and Zn are abundant in the follicular wall in both theca interna and the granulosa (Ceko et al., 2015). Trace elements especially Se and Zn are involved in cholesterol synthesis (Zheng et al., 2015). Cholesterol is the precursor for synthesizing estrogen and progesterone in human and animals. It has been reported that the relationship between trace elements and steroids is reciprocal and both act synergistically during the development and maturation of the Graffian follicle (Kumar et al., 2011).
       
The above concepts are supported by the positive association between steroids and trace element in the present study. The higher concentrations of Se and Fe in large follicles compared with other ovarian structures were reported before (Kor et al., 2013). As a result, the developing follicle shunt experiences increased hemodynamic pulses due to increasing levels of estrogen and progesterone (Abd Ellah et al., 2010). It was reported previously that serum trace elements levels increased significantly in response to higher estrogen levels, especially during the latter stages of the pre-ovulatory follicles, but then fell just before ovulation (Antunovic et al., 2002). It has been found that a positive interrelationship between serum levels of trace elements and the level of estrogen (Kumar et al., 2011). A rise in these elements was associated with extrinsic estrogen administration (Dalai et al., 2017). Researchers have found changes in plasma minerals associated with changes in plasma estrogen during the estrous cycle, following ovariectomy and during the administration of exogenous estrogen (Zheng et al., 2015). Despite the marked rise in serum trace elements during the follicular phase of the estrus cycle follicular contents of the trace elements decreased (Makhlouf et al., 2020). By synthesizing protein and metalloenzymes, the ovary synthesizes cations for ovarian development, as evidenced by a decrease in follicular fluid trace elements (Kor et al., 2013).

It can be concluded that pregnancy associates with a decrease in serum trace elements in sheep and goats. Accordingly, it is recommended that these elements should be supplemented to the animals before the intended breeding time. The positive association between trace elements and steroidogenic outcome of the mature follicles in sheep and goats should be considered when future application of reproductive biotechnology in these species.

None.

The study was conducted in accordance with the Declaration of Helsinki. The animal Care and Welfare Committee for Qassim University, Saudi Arabia, approved this study (No. 321143).

None.

Derar Refaat and Ahmed Ali have laid the concept of the research, collect samples, examined animals and wrote the manuscript; Tariq Almundarij revised the manuscript; Essam Moneim was responsible for assaying the trace elements, Tamim Hassoun helped in collecting data and clinical examination; M Zeitoun assayed steroid hormones.