It is generally known that physicochemical alterations in cervical mucus occur during erectile ovarian steroids, aiding sperm penetration. While the determination of specific parameters such as spinnbarkeit, crystallization pattern, electrical conductivity, dry matter contents, and rheological values have been utilized to predict the optimum time for cattle insemination (Joshi et al., 2017; Ondho et al., 2019; Siregar et al., 2019; Abd-El-Hafeez et al., 2020). Also, cervical mucus properties at the moment of service/insemination affect pregnancy in cows. They are influenced by alterations in periestrual hormones concentrations.
The current study's findings revealed a strong association between steroid hormones and the physical features of CM, and the probability of ovulation. The pregnancy rates in cows with <9.0, 9.0-13.5 and >13.5 cm spinnbarkeit were 33.33%, 25.00% and 85.71%, also, 9.0-13.5, 13.5-15.0 and >15.0 mS/cm electrical conductivity were 20.00%, 66.67% and 83.33%, respectively. Whereas the 1st and 2nd levels of crystallization containing typical fern patterns had 80.00 and 66.67% pregnancy rates, respectively. This result was consistent with a study by Verma et al. (2014), Joshi et al. (2017), Ondho et al. (2019) and Siregar et al. (2019).
SPK decreases with increasing progesterone, increases when estrogen concentrations are high, and peaks during ovulation (Verma et al., 2014). The 3rd level of SPK (>13.5 cm) of cervical mucus had the highest percentage of pregnancy (85.71%) compared to the other two levels (33.33% and 25.00%). These results are in accordance with Rangnekar et al. (2002) and Verma et al. (2014). Joshi et al. (2017) reported that CM of 46% of estruses ranged in 8-16 cm. The pregnancy rate in cows inseminated with CM having a high SPK value (>13.5 cm) may be attributable to the appropriate phase of estrus because SPK reaches its peak shortly before or during the ovulatory stage under the estrogen effect and then falls as progesterone concentrations rise during the luteal phase in cows (Modi et al., 2011).
Cows inseminated with a higher range (>15 mS/cm) of EC had an 83.33% pregnancy rate, which could be due to the activation motility of the uterus and spermatozoa that improve the fertilization process. Layek et al. (2013) and Verma et al. (2014) reported a positive relationship between pregnancy rate and cervical mucus EC. The small difference found in various researches could be attributable to CM lysis before conductivity measurement and methodology. Joshi et al. (2017) reported that CM had the highest number of estruses (60.00%) in the conductivity range of 13.50 - 15.00 mS/cm in cows. Under the effect of estrogen, electrolyte concentrations begin to rise during estrus, resulting in increased EC. This high amount of electrolytes in CM is essential to activate the motility of uterine and spermatozoa, and the mucus' physical state, which allows ovum-spermatozoa interaction. A positive correlation between the EC of CM and pregnancy rate in cows was observed (Bishnoi et al., 1983; Layek et al., 2013) and our findings corroborate their results. The ability of cervical mucus to be pulled into threads during estrus is attributed to the presence of big molecules in the mucus and presumably depends on the estrus to LH peak duration being delayed (Bage et al., 2002).
In the present study, 75% of pregnant cows had a TFP in CM, which could be attributable to an increase in peripheral E2 concentrations during estrus, indicating the optimal time for insemination. Rangnekar et al. (2002) and Verma et al. (2014) found almost identical results. Higher pregnancy rates (80.00% and 66.67%) were shown in cows inseminated with TFP in their CM compared to AFP (25.00% and 50.00%). The greater pregnancy rate in TFP is related to the facilitated sperm motility compared to nil or AFP (JeŽková et al., 2008). Our findings are in agreement with Kumaresan et al. (2009) and Layek et al. (2013). Crystallization constitutes a means to reach a more stable, lower energy state from a metastable solution by reducing the solute concentration (Weber, 1991). In each stage of estrus, ferning crystallization will be varied. It is linked to E2 secretion as a macro-mineral stimulus in the CM (Ondho et al., 2019). According to Yavari et al. (2009), ferning can be noticed when ovulation approaches due to increased hormone E2 produced.
Mucus criteria, especially SPK, are affected by the cervical mucus acidity and function as a non-immunogenic antibacterial in the cow's reproductive tract (Ondho et al., 2019). Lu and Morresey (2006) reported that the cervical mucus release of estrogen makes the uterus resistant to infection. The hydrolysis process of bacterial cell membranes was linked to cervical mucus containing lysozyme enzyme (LYS). LYS in CM having N-Acetyl Glucosamine is aided the hydrolysis process of bacterial cells (Chimura et al., 1993). The movement of LYS in the hydrolysis process of bacterial cells damages much of the remaining bacterial cells membrane, leaving behind CM. The cervical mucus contains substances found in bacterial cell membranes. Carbohydrates, lipids, and proteins all contribute to the structure of cell membranes. During estrus, all components of cell membrane structure, including carbohydrates, lipids, and proteins, will raise the viscosity level and SPK value of the CM. (Chimura et al., 1993).
The box plot Figure (1) showed serum TP ranged from 6.1 to 7.9 g/dl with an overall median value of 7.08 g/dl. These values were in the cow's normal range, according to Mitruka and Rawnsley (1981), who stated that the normal concentration of TP for cows is 7.56 g/dl. Siregar et al. (2019) reported that serum TP levels in the fertile and the repeat breeding Aceh cows were 6.9 and 6.6 g/dl, respectively. Our findings showed that the higher TP in serum (Figure 1) and mucus (Table 2) were observed with TFP and the greater SPK and EC values. Shiraz et al. (2010) reported that low protein levels could disrupt gonadotropin production. Therefore, the elevated protein levels identified in this study were expected to boost gonadotropin production. Manas et al. (2012) claimed that amino acids and proteins were required for the biosynthesis of gonadotropin hormones (GnRH) and LH that were responsible for the onset of ovulation. Increased gonadotropin production could lead to higher steroid concentrations in the blood. The steroid concentrations are correlated to the CM physical characteristics (Rangnekar et al., 2002).
Serum and mucus total cholesterol increased with increasing SPK and EC values and typical CRS (TFP). Cholesterol is a fatty molecule produced by the liver and circulated throughout the body (Murray et al., 2003). Murray et al. (2003) stated that cholesterol is an essential component of plasma membranes and a key component of all other steroid hormones produced in the body, such as sex hormones and corticosteroids hormones, as well as bile acids and vitamin D. Arosh et al. (1998) discovered a strong correlation between low cholesterol levels and reduced steroidogenesis concentrations. Highshoe et al. (1991) revealed that under the action of LH, cholesterol served as a precursor for the synthesis of estrogen, progesterone, and androstenedione by granulose cells. Increased steroidogenesis raises the concentration of steroids in the blood, which impacts the CM properties (Lim et al., 2014).
The current results showed the highest glucose levels in serum and cervical mucus with >13.5 cm SPK, >15 mS/cm EC, and TFP (1st & 2nd levels of CRS). The findings showed that there was clear correlation between GLU levels and tested characteristics of CM. GLU level that was decreased with reduction of SPK and EC values. TP, TC and GLU levels had a greater impact on the CM properties in this investigation. Low serum GLU levels may lead to a decline in hypothalamic GnRH release, due to a shortage of ATP, which activates cAMP as an intracellular messenger (Murray et al., 2003). The reduction in GnRH release was followed by a reduction in the of FSH and LH synthesis, which led to ovarian dysfunction or the lack of ovarian follicle growth, decrease insulin and IGF-1 concentrations, and decrease releasing of E2 by ovarian follicles (Mulligan et al., 2007).
All values of macro-minerals were in the normal range, whether in serum or mucus with all CM properties, whereas the Na concentrations were low, but K and Cl concentrations were high for the 3rd level of SPK and EC and the 1st two levels of CRS (TFP). These results were in agreement with Siregar et al. (2019). This demonstrates the specific effect of macro-minerals concentration on cervical mucus SPK, EC, and CRS. Many factors cause macro-minerals concentration to affect CM properties. Sodium chloride in the body plays an important role in reproduction, especially in the estrus stage. During estrus, sodium chloride levels are high and can cause a rise in E2 concentrations. Cervical mucus secretion is influenced by estrogen. There was a negative relationship between Na and both of K & Cl concentrations in the current results, which agreed with the findings of Abd-El-Hafeez et al. (2020), could be clarified by Coppock et al. (1982), who reported that chloride is absorbed more efficiently than sodium in the posterior section of kidney and intestine. According to some studies, Na content was the only mineral that changed significantly during the 3 days preceding estrus (Cowan and Larson, 1979). Lack of body sodium will cause ovarian dysfunction, which leads to repeated breeding (Roberts, 2004). Furthermore, high K administration in cows can cause delayed puberty and ovulation. Such high administration reduces corpora lutea development and a higher likelihood of anestrus (Velladurai et al., 2016). Ondho et al. (2019) reported that the sodium chloride percentage of CM has 2 peaks at the end of the estrus cycle. Dairy cows at younger ages (three years) reach their peak earlier than older cows while having a smaller NaCl % of CM at the peak. It was found to be related to the E2 and mineralocorticoid hormone (MCTH) secretion. NaCl % in CM from each estrus stage and age was influenced by E2 induced by LH hormone (Makmun et al., 2017; Samsudewa et al., 2019). Otherwise, high E2 secretion causes hypofyse to produce more adrenocorticotropic hormone (ACTH). Cortisol production in the liver is stimulated by increased ACTH secretion, accompanied by elevated estrogen circulation in the liver. Cortisol induces secretory MCTH as controller of electrolyte fluid in the adrenal cortex, whereas E2 can be eliminated via urine by conjugating water-soluble E2. MCTH promotes the kidneys to retain more Na+, K+, Cl−, and H+. The minerals created can be reabsorbed by the entero-hepatic together with free E2. The entero-hepatic system absorbs the minerals into each specific organ, including the cervix as one of the constituent organs of CM (Widiyono et al., 2013).
Regarding steroid hormone concentrations, there was a negative correlation between P4 and E2 in both serum and mucus. The lowest values for P4 and highest values for E2 were in cervical mucus characterized by >13.5 SPK, >15.0 EC, and typical fern pattern. P4 and E2 can influence CM physical properties (Rangnekar et al., 2002). A reduction in circulating P4 would be predicted to increase LH pulse frequency (Lopez et al., 2005). Pulse frequency on LH is raised, and LH influx is restrained when P4 maintains low concentrations causing prolonged follicular dominance (Revah and Butler, 1996). P4 takes precedence over E2 in controlling estrus behavior because P4 can impede estradiol's estrus-inducing effects (Allrich, 1994). Lopez et al. (2005) suggested that P4 concentrations in superovulated cows are greater throughout estrus than in single ovulating animals. Abnormalities in hormonal balance in the pre- and periovulatory periods have been associated with aberrant follicular/oocyte maturation (Callesen et al., 1986). Therefore, high P4 concentrations in other tested groups led to changes in CM properties.
Concerning elevation of estradiol concentrations, Lopez et al. (2005) showed a premature rise in E2 was noticead in cows with triple dominant follicles. Pre-ovulatory rise in E2 concentrations triggers the onset of LH influx (Hansel and Convey, 1983). In cattle, E2 formation from the pre-ovulatory follicle is mirrored in the serum E2 concentration, which rises gradually until the pre-ovulatory LH increase (Bevers and Dieleman, 1987). The presence of E2 has a significant impact on animal physiology throughout the estrus period and allowing the animal to copulate (Siregar et al., 2019). Tsiligianni et al. (2011) reported that in the follicular phase, superovulated cows possess the lowest and highest E2 concentration of 30.95 and 54.77 pg/ ml, respectively. The elevation in E2 concentrations in the bloodstream reaches the anterior pituitary, which increases the LH secretion. Furthermore, estrogen has an effect on the neurological system, making animals restless and causing them to mount other cows. E2 also induces a gentle contraction of the uterus, which enables spermatozoa to be transferred to the female reproductive genitals post-natural insemination. Other impacts of high E2 concentrations include blood vessels dilation in the genitals and the release of mucus by the vagina and cervical glands (Ramli et al., 2016). Properties of the mucus rely upon the hormones secreted during estrus (Benbia et al., 2011). Spermatozoa motility is aided by having mucus ducts that can easily penetrate and direct the sperm forward. This situation is related to estrogen's impact during estrus, which regulates glycoprotein macromolecules to reduce the distance among mucus molecules to 2-5 µm. Thus, a duct is formed and sperm can penetrate it (Hafez and Hafez, 2000).