Journal of Animal and Poultry Production Association among Spinnbarkeit , Electrical Conductivity , and Crystallization of Cervical Mucus and Pregnancy Rate in Egyptian Baladi Cows

Alteration of the biophysical and biochemical characteristics of cervical mucus (CM) due to differences in steroid hormones through the estrus cycle leads to different pregnancy rates. This investigation aims to study the possible relationship between CM properties with biochemical profiles, macro-mineral levels, and steroid hormones concentrations, in reference with pregnancy rates of Egyptian Baladi cows. Fourteen Baladi cows were used and synchronized. The model log-likelihood ratio was highly significant ( P =0.0009), and reported that the spinnbarkeit (SPK), electrical conductivity (EC), and crystallization (CRS) had significant effect on pregnancy rate. The 3 rd level of SPK (>13.5 cm) and EC (>15 mS/cm) showed significantly the highest effect ( P =0.0016 & 0.0517, respectively) and a clear positive of estimate marginal effect (20.2543 & 10.6192, respectively) attitude towards the pregnancy rate. However, in case of the CRS, the significant effect was in the first two levels ( P =0.0321 & 0.0425, respectively) with pregnancy rate, reverse the last 2 levels. Total proteins, cholesterol, glucose, potassium, and chloride levels, and estradiol concentrations were increased with increasing levels of SPK and EC, and appearance of typical fern patterns (first two levels of CRS), in contrast to sodium level and progesterone (P4) concentrations that decreased with elevating levels of SPK and EC and appearance of atypical fern patterns (last two levels of CRS). There was a close correlation between CM properties and steroid hormones (P 4 & E 2 ). So, alterations in CM properties, especially SPK, EC, and CRS, can be utilized to foresee estrus time and, as a result, insemination time.


Introduction
The endocervix secretory cells continuously produce cervical mucus, which varies in quality and quantity depending on the hormonal status of the estrous cycle (Lopez-Gatius et al., 1993;Tsiligianni et al., 2001). Changes in cervical mucus (CM) rheology ( ow and deformation characteristics) during the estrous cycle partially re ect the cervical epithelium's response to steroid hormones (Carlstedt et al., 1989). For instance, biophysical and biochemical properties of CM can be changed with progresses the estrous cycle of cow (Carlstedt et al., 1989;Lopez-Gatius et al., 1993). Examination of uids secreted in the female genital system to determine fertility has become a major concern and cervical uid is also involved in sperm survivability and delivery to the uterus (Kumar et al., 2012). CM characteristics can be utilized as a sign to detect the exact moment of arti cial insemination and can be utilized as an indicator of reproductive health connected to estrous behavior, also, play a major in uence in pregnancy success; thus they will boost the conception rate (Tsiligianni et  The Spinnbarkeit (SPK) value of CM is ascribed to the presence of big molecules and is thought to be dependent on molecular chain branching and other strong intermolecular forces (Elstein, 1974). The SPK achieves its greatest value around the periovulatory stage in cows (Hamana et al., 1971). SPK levels change during the periestrus stage, becoming lower as estrus progresses (Bishnoi et al., 1982). Once the SPK value has declined, the progesterone impact is started. Hormonal interventions of estrous induction also reduce SPK compared to spontaneous ovulators (Tsiligianni et al., 2001). Crystallization (CRS) reaches a maximum value near the time of ovulation (Wolf, 1977). This value reaches the peak in the pregnant cows (Tsiligianni et al., 2000). Similar to SPK, CRS is higher in spontaneous estrus cows than cows entering heat post hormonal interventions (Tsiligianni et al., 2001).
Several studies investigated the relationship between the electrical resistance property of CM and the oestrus in cows (Hulsure et al., 1995). The electrical resistance of cervical mucus (ERCM) decreases dramatically during oestrus, and cows inseminated with low ERCM had a higher conception rate than those inseminated with high ERCM (Leidl and Stolla, 1976;Ahmed et al., 2017). Layek et al. (2013) reported that SPK level and the arborization structure of cervical mucus strongly correlate with plasma progesterone (P 4 ) concentration and time of ovulation. Joshi et al. (2017) reported that the SPK level of CM ranged from 8 to 16 cm in 46% of estrus cows, and 58% of them had primary, secondary, and tertiary venation, which is typical of ferns. CM has a pH range of 7.5-8.0 and a conductivity of 13.50-15.00 mS/cm.
We suffer a big problem in the baladi cows, where the movement is large and increasing in a way that makes it di cult for him to follow the details of the occurrence of estrus features with certainty and thus determine the most appropriate time for insemination compared to the foreign breeds, with which it is easy to follow in detail the appearance of the occurrence of estrus and determine the most appropriate time for vaccination. Therefore, observing the physical characteristics may be a strong alternative to determine the most appropriate time for insemination in baladi cows. The objective of this study is to nd out possible relationships among SPK, EC and CRS of cervical mucus and pregnancy rate in Egyptian Baladi cows.

Animals
Fourteen Egyptian Baladi cows were selected in good body condition and normal anatomy genitalia. Cows had the same parity, aged 4-5 years and weighing 400 ± 11.35 kg. which had already calved and had undergone at least two regular cycles, and then were synchronized by an intramuscular injection of 25 mg prostaglandin-F2α. Cows were fed according to NRC (2001) and were housed in a semi-open shade with ventilation and a sprinkler system to keep them cool. All cows were reared at the Sids Experimental Station belonging to the Animal Production Research Institute (APRI), Agricultural Research Center (ARC), Ministry of Agriculture and Land Reclamation, Egypt. The animal care and use committee from the Animal Production Research Institute approved our study.

Cervical mucus (CM) and blood samples
All cows were observed twice a day for 30 minutes to detect estrus. Samples of CM and blood were immediately collected prior to natural insemination. The cow's vulva was cleaned with an antiseptic solution and swilled by distilled water and properly wiped. A exible plastic sheath was utilized to collect CM samples to avoid vaginal mucosa rupture. A sterile syringe (10 ml) was a xed to the sheath's exterior end. The samples were gently suctioned from the cervical aperture and surrounding area before being kept at -20°C for future use.
CM samples were immediately checked for spinnbarkeit (SPK), electrical conductivity (EC), and crystallization (CRS). The SPK value was measured using a simple apparatus, as described by Tsiligianni et al. (2000). Brie y, a small drop of mucus was placed on a glass slide and covered with a glass coverslip. The SPK was determined by drawing the mucus out vertically until the mucus thread broke.
This procedure was repeated twice for each sample, and the mean value was recorded and categorized into three groups (<9, 9-13.5 and >13.5 cm).
Before performing the EC measurement, CM samples were rst stirred in a mixture vortex (Spinix Corporation, CA, USA) until they were lysed and turned into a free-owing liquid. Then EC was determined in the lysed CM utilizing a pH-Conductivity Benchtop (Orion 4 star, Thermo Electron Corporation, USA) in mili-Siemens/cm unit (mS/cm) and split into three groups (9-13.5, 13.5-15 and >15 mS/cm). CRS was measured as described by Tsiligianni et al. (2000). Smearing a drop of CM on a clean slide was used to prepare a lm. The lm was left to dry for 30 minutes at 25°C before being inspected under 40× magni cation. CRS pattern was rated on a scale of 1 to 4; 1: typical crystals formation or typical fern patterns (TFP); 2: Formation of many typical and few atypical fern-like crystals; 3: Formation of many atypical fern-like crystals and few typical ones; and 4: Formation of only atypical fern patterns (AFP). Mucus specimens were stored at -20°C until the chemical analysis of total protein (TP), total cholesterol (TC) and glucose (GLU), sodium (Na), potassium (K), chloride (Cl), progesterone (P 4 ) and estradiol (E 2 ).
Blood serum was taken from cows' jugular veins using a 10 ml disposable syringe. Blood was collected just before insemination.
Serum and mucus TP, TC, and GLU analysis were carried out by spectrophotometer using a commercial kits (Spinreact, Spain). Na and K concentration were determined by colorimetric and turbidimetric methods, respectively, using commercial kits (Biodiagnostic Company, Giza, Egypt). Cl concentration was determined using a thiocyanate method (QCA Company, Amposta (Tarragona), Spain).
The progesterone (P 4 ) concentrations were determined by using a commercial RIA kit (Coat-A-Count; Diagnostic Products Corporation, DPC, Los Angeles, California, USA) and the estradiol (E 2 ) was purchased from Spectria1; Orion Diagnostica Oy (Espoo, Finland).

Statistical analysis
The data of cervical mucus properties (spinnbarkeit, electrical conductivity, and crystallization) were transformed by the ARCSine method (Kirk, 2013) to calculate the correlation coe cient among physical (SPK, EC, & CRS) and chemical (TP, TC, GLU, Na, K & Cl) properties of cervical mucus, as well as steroid hormones by using SAS (2014). Data were statistically analyzed using the general linear model procedure. The differences among means were tested using Duncan's Multiple-rang test.
Box-and-whisker plots (whiskers are 1.5× the interquartile range) for each group (SAS, 2014) were used to analyze physical properties data. Box plots display batches of data are becoming a widely used tool in exploratory data analysis (Mcgill et al., 1978).
A logistic regression using the GENMOD procedure of SAS (2014) was performed to assess the signi cance of cervical mucus properties (spinnbarkeit, conductivity, and crystallization).
The following model was used to determine cervical mucus properties affecting pregnant in Egyptian cows: The previous model was applied also to study the factors in Egyptian cows.

Results
Out of fourteen cows presented in Table (1), three and four cows had mucus with <9.0 cm and 9-13.5 cm SPK, respectively, only one of them became pregnant, and seven cows had mucus with >13.5 cm SPK, six became pregnant. Moreover, ve cows had mucus with 9.0-13.5 mS/cm EC, only one became pregnant, three cows had mucus with 13.5-15 mS/cm EC, two became pregnant, and six cows had mucus with >15 mS/cm, ve of them became pregnant. However, ve and three cows had mucus with TFP (1st & 2nd levels of CRS, respectively), only one of them did not get pregnant; also, ve and one had mucus with AFP (3rd & 4th levels of CRS, respectively), only one of them get pregnant.
Pearson correlation coe cient by ARCSine method between properties of cervical mucus (SPK, EC & CRS) and studied parameters (biochemical, macro-minerals & steroid hormones) are presented in Table  (5). SPK and EC were positively (P < 0.01 & 0.001) correlated with the concentrations of TP, TC, GLU, K, CL and E 2 . In contrast, the crystallization had an inverse (P < 0.001) relationship with the previous parameters. An opposite trend was observed with Na levels, and P 4 concentrations.

Discussion
It is generally known that physicochemical alterations in cervical mucus occur during erectile ovarian steroids, aiding sperm penetration. While the determination of speci c parameters such as spinnbarkeit, 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 ndings 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 E 2 concentrations during estrus, indicating the optimal time for insemination. Rangnekar et al. 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 ndings 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 identi ed 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).  2020), could be clari ed by Coppock et al. (1982), who reported that chloride is absorbed more e ciently than sodium in the posterior section of kidney and intestine. According to some studies, Na content was the only mineral that changed signi cantly 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 (Hansel and Convey, 1983). In cattle, E 2 formation from the pre-ovulatory follicle is mirrored in the serum E 2 concentration, which rises gradually until the pre-ovulatory LH increase (Bevers and Dieleman, 1987). The presence of E 2 has a signi cant impact on animal physiology throughout the estrus period and allowing the animal to copulate ( 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).

Conclusion
According to the ndings of this study, alterations in cervical mucus properties, especially SPK, EC, and CRS, can be utilized to foresee estrus time and, as a result, insemination time. Cows with > 13.5 cm SPK and > 15 mS/cm EC of their CM showed the highest pregnancy rate and higher percentage cows. The typical fern pattern was seen in a higher percentage of cows' CM, mainly composed of primary and secondary venation. Further studies should be conducted to explain the heterogeneity of mucus secretion in cows upon estrus synchronization. These ndings may also help to update or change insemination practices to get a higher pregnancy rate by inseminating at an accurate periovulatory time in some cow breeds raised in similar climate condition, or similar reproductive characteristics.