- Email: [email protected]
or PGF2α
ELSEVIER
CHEMICAL PROPERTIES OF BOVINE CERVICAL MUCUS DURING NORMAL ESTRUS AND ESTRUS INDUCED BY PROGESTERONE AND/OR PGF2a Th. Tsiligianni,’
A. Karagiannidis,’
P. Brikas,’ and Ph. Saratsis’
‘Clinic of Obstetrics and Al and ‘Laboratory of Physiology Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Received for publication: Accepted:
June
Greece
23, 2000
January 24, 2001
ABSTRACT Ninety-two Friesian cows were used to determine the chemical properties of cervical mucus during normal estrus and estrus induced by progesterone (P&releasing intravaginal devices (PRID) and/or prostaglandin F2a. The animals were assigned to 4 groups (no treatment, a PRID for 12 days plus injection of 1000 IU PMSG at the removal of PRID, a double im injection of PGF2, 11 days apart, or PRID for 7 days plus an im injection of PGF2, 24 h before the removal of PRID). A number of cows with normal estrus exhibited three consecutive estrous cycles after delivery. Cows that had not shown estrus for 3 months after delivery had their ovaries palpated twice at IO-day intervals, to determine their ovarian activity. Then PRID and/or PGF2a was administered in cows that had a palpable corpus luteum in one of the two palpations (cyclic cows). A double artificial insemination (Al) was performed to the cows of the three inducedestrus groups, while the cows with normal estrus received only one Al. Cervical mucus samples were collected from all cows 5 to 30 min before the first Al. Additionally, samples of cervical mucus were collected from 20 cows during their first estrus after the induced one. The results are summarized as follows: 1) The biochemical properties of cervical mucus in the first three estrus periods after delivery were similar. 2) These properties were similar both in normal estrus after delivery and in the first estrus after an induced one. 3) Glucose and fructose concentrations for normal estrus were similar to those for induced estrus groups. 4) Total protein and cholesterol concentrations were significantly lower (P c 0.001) in normal than in induced estrus, while no difference was found among the induced estrus groups. 5) Pregnancy rates of the cows did not differ significantly among the normal and the induced-estrus groups. 6) The percentages of cows in the induced-estrus groups that produced cervical mucus with total protein and cholesterol concentrations similar to those for the normal estrus groups was very low. 8 2001 by Elsevier Science Inc. Key words: cervical mucus, biochemical properties, cow, estrus, normal, induced INTRODUCTION Cervical mucus contains about 92 to 95Oh water. During e&us, when mucus is most abundant, water content rises and this situation is known as water phase (2,13). Components of cervical mucus, besides cellular elements, are semisolid gel constituents and a fluid regarded as cervical plasma (14). Low molecular weight Theriogenology 56:41-W 8 2001 Elsevier Science
2001 Inc.
0093-691WOl@-see front matter PII: SOO93-691X(01)00541-6
42
Theriogenology
such as electrolytes, carbohydrates, amino acids, lipids and soluble macromolecular compounds such as soluble and locally produced proteins and polysaccharides are dissolved in it (19). The structure elements of the mucus gel consist, according to Gibbons (1959) of a polydisperse system of long glycoprotein molecules with a high degree of branching. Certain blood serum proteins have been identified in cervical mucus (19). Proteins in cervical mucus derive partly from the blood serum and are partly produced somewhere in the genital tract (4). The way proteins deriving from the serum enter the mucus is unknown. A number of carbohydrates have been identified in cervical mucus. The most important of them are glucose and fructose. These compounds appear to be the major free sugars in uterine flushings from the non-pregnant rat, rabbit, cow, ewe and pig (5,9,23,31). Lipids may play a nutritive role for spermatozoa, and there is speculation that they may also protect spermatozoa from temperature changes (12). Cholesterol, which is the precursor of the steroid hormones (24) is related to the stages of the estrous cycle (29). The purpose of the present cervical mucus in cows at either and/or PGF2a and to determine induced estrus, at the time of first
research was to study the biochemical properties of normal estrus or induced estrus using progesterone whether these properties are different in cows with Al.
MATERIALS AND METHODS Animals This study was conducted from September 1995 to December 1996 at five farms in Northern Greece. Ninety-two Friesian cows of different ages (2.5 to 7 years old) and parities were used. Feeding and housing conditions were similar in all five farms. All cows were healthy and free of abnormal genital discharges and anatomical abnormalities of the reproductive tract. Cows included in this study where either observed in estrus after the last calving or had not shown estrus for three months after calving. In the latter case, cows had their reproductive system palpated twice at IO-day intervals, to determine their ovarian activity. These cows were cyclic with ovarian activity confirmed by the presence of a palpable corpus luteum at one of the two palpations. The cows were assigned to one of the following four groups: Normal estrus. Forty-one cows that were observed one to three times in estrus after their last calving, received no treatment, serving as the group of cows with normal spontaneous estrus. Sixteen of these cows exhibited three consecutive estrous cycles with a range of 19-21 days. No cow of that group were induced in estrus. P,-induced estrus. In 25 cyclic cows that had not shown overt estrus for three months after their last calving, estrus was induced with a Progesterone (Pd)-Releasing lntravaginal Device (PRID - Abbovestrol@, Ceva, France) for 12 days plus 1000 IU im of Pregnant Mare Serum Gonadotrophin (PMSG - Intergonan”, Intervet, Netherlands) at
Theriogenology
43
PRID removal. Seven animals were excluded from the trial because of the inadequate quantity of cervical mucus collected (4 cows) or loss of the PRID (3 cows). PGF2a-induced estrus. In 20 cyclic cows that had not shown overt estrus for three months after their last calving, estrus was induced with a double im injection of PGF2a (Gabbrostim@, Vetem, Italy), 11 days apart. Three of these cows were excluded because of the inadequate quantity of cervical mucus collected. PA+ PGF2ainduced estrus. In 20 cyclic cows, which had not shown overt estrus for three months after their last calving, estrus was induced with a PRID (without the EZ benzoate capsule) for 7 days and an im injection of PGF2a one day before the PRID removal . Four of these cows were excluded from the trial because of the inadequate quantity of cervical mucus collected (3 cows) or loss of the PRID (1 cow). Collection of Cervical Mucus Cervical mucus samples were collected from all cows 5 to 30 min before the single Al in the cows with normal estrus and before the first Al in the cows with induced estrus. Specifically, samples were collected after estrus signs were observed and before the end of estrus in cows with non induced estrus, 24 h after the removal of PRIDs, in Pq and Pq+ PGF2a-induced estrus and 48 h after the second injection of PGF2a. The vulva of the cow was washed with disinfectant solution, rinsed with water and then dried. Cervical mucus samples were collected using a plastic pipette 10 mL wide and 35 cm long (26). The front end of the pipette was cut at 0.5 cm length to make the opening wider and was smoothed to avoid wounding the genital tract. At the other end of the pipette, a 1m plastic, transparent tube was attached. Cervical mucus was collected from the external OS of the cervix and the area around it by means of oral aspiration. Cervical mucus was placed to a glass tube of 10 mL and transported to the laboratory in a refrigerated box (4“ C) and used within 2 hours after its collection. Aliquots of 1.5 to 2 mL of cervical mucus were homogenized in a Junk and Kunkel homogenizer and stored at -30’ C for future use in measurement of biochemical properties. Artificial Insemination After collecting the cervical mucus samples, all cows were inseminated with frozen semen of proven fertility, once the cows with normal estrus which presented estrus signs and twice the cows with induced estrus, 24 h and 48 h after the removal of PRlDs or 48 h and 72 h after the second dose of PGF2a. In the induced estrus groups, cows were not observed for estrus signs and double blind Al was performed. Spermatozoa were placed at the last third of the cervix for all cows. Pregnancy was determined by palpation per rectum 3 months after insemination. Biochemical Analysis Total protein. Total protein concentration in cervical mucus homogenate was determined by the method of Lowry et al. (8) as modified by Peterson (15). The same method was used by Prasad et al. (16). For this purpose a Sigma kitTUfor total protein measurement was used. The standard curve was done using total protein standard
44
Theriogenology
solution (400 vg purified bovine serum albumin /mL) and an aliquot of 0.1 mL homogenized cervical mucus were used. Results were expressed in pg/mL. Glucose. Glucose concentration was measured by the GOT-PERID method (25) using Boehringer reagents. Glucose standard solution (500mg/lOOmL) and an aliquot of 0.1 mL homogenized cervical mucus were used. Results were expressed in mg/lOO mL. Fructose. Fructose concentration was measured by the method of Mann (10) using Merk reagents. Fructose standard solution (100 ug/mL) and an aliquot of 0.2 mL homogenized cervical mucus were used. Results were expressed in pg/mL. Cholesterol. Cholesterol concentration was measured by the GOD-PAP method (20,21,25) using the kit of Elitech, that contains two reagents and a standard (Reagent 1: Pipes buffer, pH 6.90 SOmmollL, Phenol 24mmoVL and Sodium cholate O.Smmol/L. Reagent 2: Cholesterol esterase 2 200 U/L, Cholesterol oxidase 2 250 U/L, Peroxidase 2 1000 U/L and 4-Aminoantipyrine 0,5 mmol/L.)The same method used by Zaaijer et al. (29). Cholesterol standards in a range between 0.005 and 0.500 mmol/L and an aliquot of 0.1 mL homogenized cervical mucus were used. Results were expressed in mmol/L. Biochemical assays used in this study have been used by other researchers; accuracy, detection limits and quantification limits were acceptable. Statistical Analysis Analysis of variance (Anova) was used to compare cervical mucus biochemical properties among normal and induced-estrus groups. Comparisons between means were performed by Dunkan’s new multiple range test (22). Distribution was normalized by the proper transformation (logarithmic for total protein and fructose and reversal for glucose) where needed. The results were expressed as mean f SEM. A paired sample t-test was used to compare cervical mucus biochemical properties among the first three consecutive normal estrus cycles after delivery. T-test for independent samples was used to compare cervical mucus characteristics between normal estrus and the first estrus after the induced estrus. Chi square analysis was used to compare pregnancy rates among normal and induced-estrous groups and to estimate the percentages of cows in the induced-estrous groups that produced cervical mucus with similar physical properties to those of the normal estrous group. All analyses were performed using the statistical software program SPSS RESULTS Data showing biochemical properties of cervical mucus during three consecutive normal estrus periods after delivery are shown in Table 1. There were no differences in the biochemical properties of cervical mucus among the 15’, 2”d and 3’d estrus. Data showing biochemical properties of cervical mucus in the normal estrus after delivery (41 cows, 73 samples) and the first estrus after induction (20 cows, 20 samples) are shown in Table 2. There were no differences between these two groups. Therefore, all 61 cows constituted the normal estrus group.
45
Theriogenology
Table 1. Biochemical properties of cervical mucus (mean*SEM) in the first three consecutive normal estrus after delivery of 16 cows Cholesterol Fructose protein Glucose Total (mmol/L) (mg/lOO mL) (pg/mL) (pg/mL) Estrus 0.11 Ito. 5.2f0.7 3.6i2.2 1272.9*415.4 First 0.19*0.11 2167.4*1022.9 6.1*0.7 5.9*2.9 Second 0.11*0.05 17.2*10.9 1639.1*490.5 9.2*5.6 Third There were no statistically significant differences among groups. Table 2. Biochemical properties of cervical mucus (mean*SEM) in the normal estrus and in the first estrus after induction Estrus NED (41/73) FEI (20/20)*
Total protein (pg/mL) 1379.1 i153.7 1036.9*179.6
Glucose (mg/lOOmL) 7.7ti.2 4.9*0.9
Fructose (ug/mL) 4.6*1.4 5.9i4.1
NED: normal estrus after delivery FEI: first estrus after induction * Number of cows/number of samples used for the determination properties. There were no statistically significant differences between groups.
Cholesterol (mmol/L) 0.09*0.02 0.07&0.04
of biochemical
Data showing biochemical properties of cervical mucus in the normal (61 cows, 93 samples) and in the induced-estrus groups are shown in Table 3. The treatment used to induce estrus had an effect on total protein and cholesterol concentrations. Total protein concentrations and cholesterol concentrations were significantly lower in the normal-estrus group compared to those in the three induced-e&us groups (P c 0.001). Glucose and fructose concentrations were similar in the normal and in the inducedestrus groups. Table 3. Biochemical properties of cervical mucus (mean f SEM) in the normal and in the induced estrus groups Estrus group Normal (61193)’ Induced by Pq (18/18)* PGF2a (17/I 7)* P4+PGF2a (16/I 6)*
Total protein (ug/mL) 1330.2” f 134,5
Glucose Fructose (mg/l OOmL) (pg/mL) 7.3Of 1.9 4.ga* 1.3
Cholesterol (mmol/L) 0.09” f 0.02
6923.7b * 1631.5 6485.gb * 1740.7 5498.4b f 2006.8
16.O’f 3.2 8.3” * 1.7 11.7’ *3.2
0.87b f 0.25 0.65b * 0.21 0.86b f 0.40
12.3’ f 5.6 14.1’ * 5.6 6.1a *4.3
()* Number of cows/number of samples used for the determination of biochemical properties a,b’c Columns with different superscripts differ P
46
Theriogenology
Data showing pregnancy rates for normal and induced-estrus groups are shown in Figure 1. There were no differences in pregnancy rates among these groups, Finally, data showing percentages of cows in the induced-estrus groups with similar biochemical properties (mean&EM) to that in the normal estrus are shown in Table 4. Only a small percentage of cows in induced-estrus groups produced at the day of first Al cervical mucus had biochemical properties similar to those in the normalesJus group.
0 60
0
Normal Induced by
Normal
m
p4
m
PGFza
m
Pb+PGFza
\
estrus
I Induced estrus
Figure 1. Pregnancy rates in the normal and in the induced estrus groups. There were no statistically significant differences among groups. Table 4. Percentages of cows with similar biochemical properties (mean*SEM) in normal and induced-estrus groups Normal Biochemical properties
p4
meankSEM Total protein II 330.2k134.5
Wmb
5.9
n=18 mean&SE F232.6)
30.8
(758.5k243.20.0
(770.2k336.3)
5)
Glucose < 7.3zt1.9 (mgll OOmL) Fructose I4.9k1.3
(ug/mL) Cholesterol
%
Estrus induced by PGF2a P4+PGF2a n=16 n=17 % mean&EM Oh mean&EM
I 0.09*0.02
50.0
(5.6k0.9)
70.6
(4.5k0.7)
66.7 (5.820.8)
70.6
(I. 1kO.6)
66.7
(0.9kO.5)
84.6 (0.4*0.4)
22.2
(0.05*0.02)35.3
(0.06*0.01)
60.0 (0.04&0.01)
Theriogenology
DISCUSSION In the present study, it has been found that biochemical properties of cervical mucus collected in the first three consecutive normal estrus cycles after delivery do not differ significantly. Biochemical properties of cervical mucus collected during the first estrus after the induced one also do not differ from those of normal estrus. Therefore, the first estrus after the induced could be considered a normal one in terms of biochemical properties. However, only total protein and cholesterol concentrations differ in the normal and in the induced-estrus groups. On the contrary, glucose and fructose concentrations did not differ. Additionally, the percentages of cows in the induced-estrus groups producing at the time of the first Al cervical mucus with total protein and cholesterol concentrations similar to those in the normal-estrus group, were very low. Furthermore, cervical mucus collected from non induced estrus was abundant, clear and transparent, while that from most induced-estrus cows was thicker and less abundant. Total protein concentration in the cervical mucus collected from cows with PGF2ainduced estrus was similar to that observed by Prasad et al. (16). Furthermore, Prasad et al. (16) found that total protein concentration is higher (almost double) in cervical mucus collected the day before estrus manifestation than that collected during the day of estrus in the PGF2a-induced estrus group. They observed lower total protein concentrations on the day of estrus. They also observed increased proteins in cervical mucus collected from cows that did not exhibit estrus signs, as well as in cervical mucus collected from controls during Day 12 of the estrous cycle (16). In the present study, total protein concentration in the PGF2a-induced estrus group was similar to that in normal estrus for only 30.8W of cows at the day of first Al. Total protein concentration could be reduced when the second Al was performed. However, this suggestion must be studied further. Additionally, Rexroad and Barb (17) observed that total protein concentration was higher in the cervical mucus of ewes when estrus was induced by progesterone-impregnated sponges than it was in the control group, but it was lower in cervical mucus of ewes that had received MAP than it was in the control group. Cholesterol concentration in the cervical mucus of cows with normal estrus is similar to that observed by Zaaijer et al. (29). Contrary to Zaaijer et al. (29) who found lower cholesterol concentration in cervical mucus collected from cows with progesterone (PRID) and prostaglandin F2a-induced estrus compared to the results of the present work. Additionally, Zaaijer et al. (29) found similar cholesterol concentration in cows with normal and with induced estrus, but estrus was exhibited 72 h after PRID removal, that is, when a second Al was performed in the present study. It is supposed that in the present study cholesterol concentration might be reduced during the second Al. In the present work, 60% of cows with P4+ PGF2a-induced estrus had cholesterol concentrations similar to those of cows with normal estrus, and the mean value is similar to that reported by Zaaijer et al. (29). Glucose and fructose concentrations of cervical mucus do not differ significantly in cows with normal and induced estrus. On the contrary, El-Naggar and Horrath (3) found higher fructose concentration in the cervical mucus of cows with normal estrus than that observed in the present study, but the method they used for determination of fructose was criticized by Mann (11). Furthermore, the fructose concentration found in the
48
Theriogenology
present study cannot be compared to that found in other studies (27) because of the different determination methods used. Glucose concentrations in our study were lower than those reported by Weed and Carrera (28) in the cervical mucus of women. On the contrary, Bane and Rajakoski (1) merely traced glucose in the estrous cervical mucus of cow, using calorimetric tips, while Zaaijer and Van der Host (30) observed differences in glucose concentration depending on ovulation and conception. Furthermore, glucose and fructose concentrations found in the present study were significantly decreased in a high percentage of cows with induced estrus on the day of first Al. The reason for that must be investigated. Despite the different total protein and cholesterol concentrations of cervical mucus among normal and induced estrus groups, pregnancy rates did not differ significantly among these groups. Therefore, biochemical properties of cervical mucus do not directly affect fertility. In the induced-oestrus groups a second Al was performed 24 h after the first one. Biochemical properties of cervical mucus could be similar to those in normal estrus at the time of second Al. Furthermore, the percentages of cows in the induced-estrus groups producing at the time of the first Al have similar cervical mucus as in the normal estrus are very low. The biochemical properties that mostly differ are the total protein and the cholesterol concentrations. It has been found that cows do not exhibit estrus at a determined time (6,18). In summary, the results from the present study showed that biochemical properties of cervical mucus are similar during the first three consecutive normal estrus periods after delivery; that the first estrus after an induced one can be considered as a normal estrus; that total protein and cholesterol concentrations are significantly different in the normal and in the induced-estrus groups; that a small percentage of cows with induced estrus produces at the day of first Al cervical mucus with concentrations of total protein and cholesterol similar to those for cows with normal estrus. These two properties could be used as indicators of the optimal time for Al after induced estrus. REFERENCES 1.
2. 3.
4. 5. 6.
Bane A, Rajakoski E. The bovine estrus cycle. Cornell Vet 1961;51:77-95. Bergman P. Sexual cycle, time of ovulation, and time of optimal fertility in women. Acta Obst Gynec Scandinav 1960;29(Suppl. IV):6-139. El-Naggar MA, Hornath EB. Sugar content of the cervico-vaginal mucus of cattle during the sexual cycle with special reference to fructose. Acta Vet Hung 1971;21:15-20. Elstein M. The cervix and its mucus. In Clinics in Obstetrics and Gynecology. Vol 1, No 2, 1974: 345-368. Haynes NB, Lamming GE. The carbohydrate content of sow uterine flushings. J Reprod Fertil 1967;14:335-337. Garcia-Winder MJ, Gallegos-Sanchez J. Estrus synchronization in Holstein cows using reduced doses of prostaglandin Fza. Theriogenology 1991;36: 191199.
Theriogenology
7. 8. 9. 10.
11.
12. 13. 14. 15. 16.
17. 18. 19. 20. 21. 22. 23.
24. 25. 26.
27.
28. 29.
30.
49
Gibbons RA. Chemical properties of two mucoids from bovine cervical mucin. Biochem J 1959;73:209-225. Lowry CH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin Phenol reagent. J Biol Chem 1951;193: 265275. Lutwak-Mann C. Some properties of uterine and cervical fluid in the rabbit. Biochim Biophys Acta 196258: 637-639. Mann T. Studies on the metabolism of semen. 3. Fructose as a normal constituent of seminal plasma. Site of formation and function of fructose in semen. Biochem J 1946;40:481. Mann T. Energy requirements of spermatozoa and the cervical environment. In The Biology of the Cervix. Eds. R J Blandau and KS Moghissi. University of Chicago Press. 1973:334. Marcus SL. The cervical factor in infertility. J Reprod Med 1969;3:138-142. Moghissi KS. The function of the cervix in fertility. Fertil Steril 1972;23:295-306. Odeblad E. The functional structure of human cervical mucus. Acta Obstet Gynecol Stand 1968;47 (Suppl. 1):57-64. Peterson GL. A simplification of the protein assay method of Lowry et al. Which is more generally applicable. Anal Biochem 1977;83:346-356. Prasad A, Kalalyan NR, Bachlaus NK, Arora RC, Pandey RS. Biochemical changes in the cervical mucus of buffalo after induction of oestrus with prostaglandin FZaand cloprostenol. J Reprod Fertil 1981;62:583-587. Rexroad CE, Barb CR. Cervical mucus in estrous ewes after treatment with estrogen, progestogens and intrauterine devices. J Anim Sci 1977;44: 102-l 05. Roche JF, Ireland J, Mawhinney S. Control and induction of ovulation in cattle. J Reprod Fertil 1981;30 (Suppl): 21 l-222. Schumacher GFB. Biochemistry of cervical mucus. Fertil Steril 1970;21:697705. Siedel J, et al. J Clin Chem Clin Biochem 1981;19:838. Stahler F, et al. Med Lab 1977;30:29. Steel RGD, Torrie JM. Principles and Procedures in Statistics. New York McGraw-Hill. 1980 Suga T, Masaki J. Studies on the uterine secretion of the cow. 6. Sugar and polyol constituents in the luminal fluid of bovine uterus. Jap J Anim Reprod 1973; 18: 143-I 47. Trakatellis A. Cholesterol metabolism. Biochemistry. 1986:559-576. Trinder P. Ann Clin Biochem 1969;6:24. Tsiligianni Th, Karagiannidis A, Brikas P, Saratsis Ph. Relationship between certain physical properties of cervical mucus and fertility in cows. Dtsch Tierarztl Wochenschr 2000; 107 (1):28-31. Van der Linden PJQ, Kets M, Gimpel JA, Wiegerinck MAHM. Cyclic changes in the concentration of glucose and fructose in human cervical mucus. Fettil Steril 1992;57: 573-577. Weed JC, Carrera AE. Glucose content of cervical mucus. Fertil Steril 1970;21:866-872. Zaaijer D, Counotte GHM, Sol J, Smidt WJ, Broadbent PJ. Changes in the composition of cervical mucus of the cow during the estrous cycle as parameters for predicting potential fertility. Theriogenology 1993;39:569-580. Zaaijer D, Van der Horst CJG. Cyclical changes in hormones, carbohydrates and indole metabolism in cervical mucus of normal, fertilizing cows and the relationship with non-fertility. Cytobios 1983;37: 113-I 27.
50
31.
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Zavy MT, Clark WR, Sharp DC, Robberts RM, Bazer FW. Comparizon of glucose, fructose, ascorbic acid and glucose phosphate isomerase enzymatic activity in uterine flushings from non-pregnant and pregnant gilts and pony mares. Biol Reprod 1982;27:1147-1158.
CHEMICAL PROPERTIES OF BOVINE CERVICAL MUCUS DURING NORMAL ESTRUS AND ESTRUS INDUCED BY PROGESTERONE AND/OR PGF2a Th. Tsiligianni,’
A. Karagiannidis,’
P. Brikas,’ and Ph. Saratsis’
‘Clinic of Obstetrics and Al and ‘Laboratory of Physiology Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Received for publication: Accepted:
June
Greece
23, 2000
January 24, 2001
ABSTRACT Ninety-two Friesian cows were used to determine the chemical properties of cervical mucus during normal estrus and estrus induced by progesterone (P&releasing intravaginal devices (PRID) and/or prostaglandin F2a. The animals were assigned to 4 groups (no treatment, a PRID for 12 days plus injection of 1000 IU PMSG at the removal of PRID, a double im injection of PGF2, 11 days apart, or PRID for 7 days plus an im injection of PGF2, 24 h before the removal of PRID). A number of cows with normal estrus exhibited three consecutive estrous cycles after delivery. Cows that had not shown estrus for 3 months after delivery had their ovaries palpated twice at IO-day intervals, to determine their ovarian activity. Then PRID and/or PGF2a was administered in cows that had a palpable corpus luteum in one of the two palpations (cyclic cows). A double artificial insemination (Al) was performed to the cows of the three inducedestrus groups, while the cows with normal estrus received only one Al. Cervical mucus samples were collected from all cows 5 to 30 min before the first Al. Additionally, samples of cervical mucus were collected from 20 cows during their first estrus after the induced one. The results are summarized as follows: 1) The biochemical properties of cervical mucus in the first three estrus periods after delivery were similar. 2) These properties were similar both in normal estrus after delivery and in the first estrus after an induced one. 3) Glucose and fructose concentrations for normal estrus were similar to those for induced estrus groups. 4) Total protein and cholesterol concentrations were significantly lower (P c 0.001) in normal than in induced estrus, while no difference was found among the induced estrus groups. 5) Pregnancy rates of the cows did not differ significantly among the normal and the induced-estrus groups. 6) The percentages of cows in the induced-estrus groups that produced cervical mucus with total protein and cholesterol concentrations similar to those for the normal estrus groups was very low. 8 2001 by Elsevier Science Inc. Key words: cervical mucus, biochemical properties, cow, estrus, normal, induced INTRODUCTION Cervical mucus contains about 92 to 95Oh water. During e&us, when mucus is most abundant, water content rises and this situation is known as water phase (2,13). Components of cervical mucus, besides cellular elements, are semisolid gel constituents and a fluid regarded as cervical plasma (14). Low molecular weight Theriogenology 56:41-W 8 2001 Elsevier Science
2001 Inc.
0093-691WOl@-see front matter PII: SOO93-691X(01)00541-6
42
Theriogenology
such as electrolytes, carbohydrates, amino acids, lipids and soluble macromolecular compounds such as soluble and locally produced proteins and polysaccharides are dissolved in it (19). The structure elements of the mucus gel consist, according to Gibbons (1959) of a polydisperse system of long glycoprotein molecules with a high degree of branching. Certain blood serum proteins have been identified in cervical mucus (19). Proteins in cervical mucus derive partly from the blood serum and are partly produced somewhere in the genital tract (4). The way proteins deriving from the serum enter the mucus is unknown. A number of carbohydrates have been identified in cervical mucus. The most important of them are glucose and fructose. These compounds appear to be the major free sugars in uterine flushings from the non-pregnant rat, rabbit, cow, ewe and pig (5,9,23,31). Lipids may play a nutritive role for spermatozoa, and there is speculation that they may also protect spermatozoa from temperature changes (12). Cholesterol, which is the precursor of the steroid hormones (24) is related to the stages of the estrous cycle (29). The purpose of the present cervical mucus in cows at either and/or PGF2a and to determine induced estrus, at the time of first
research was to study the biochemical properties of normal estrus or induced estrus using progesterone whether these properties are different in cows with Al.
MATERIALS AND METHODS Animals This study was conducted from September 1995 to December 1996 at five farms in Northern Greece. Ninety-two Friesian cows of different ages (2.5 to 7 years old) and parities were used. Feeding and housing conditions were similar in all five farms. All cows were healthy and free of abnormal genital discharges and anatomical abnormalities of the reproductive tract. Cows included in this study where either observed in estrus after the last calving or had not shown estrus for three months after calving. In the latter case, cows had their reproductive system palpated twice at IO-day intervals, to determine their ovarian activity. These cows were cyclic with ovarian activity confirmed by the presence of a palpable corpus luteum at one of the two palpations. The cows were assigned to one of the following four groups: Normal estrus. Forty-one cows that were observed one to three times in estrus after their last calving, received no treatment, serving as the group of cows with normal spontaneous estrus. Sixteen of these cows exhibited three consecutive estrous cycles with a range of 19-21 days. No cow of that group were induced in estrus. P,-induced estrus. In 25 cyclic cows that had not shown overt estrus for three months after their last calving, estrus was induced with a Progesterone (Pd)-Releasing lntravaginal Device (PRID - Abbovestrol@, Ceva, France) for 12 days plus 1000 IU im of Pregnant Mare Serum Gonadotrophin (PMSG - Intergonan”, Intervet, Netherlands) at
Theriogenology
43
PRID removal. Seven animals were excluded from the trial because of the inadequate quantity of cervical mucus collected (4 cows) or loss of the PRID (3 cows). PGF2a-induced estrus. In 20 cyclic cows that had not shown overt estrus for three months after their last calving, estrus was induced with a double im injection of PGF2a (Gabbrostim@, Vetem, Italy), 11 days apart. Three of these cows were excluded because of the inadequate quantity of cervical mucus collected. PA+ PGF2ainduced estrus. In 20 cyclic cows, which had not shown overt estrus for three months after their last calving, estrus was induced with a PRID (without the EZ benzoate capsule) for 7 days and an im injection of PGF2a one day before the PRID removal . Four of these cows were excluded from the trial because of the inadequate quantity of cervical mucus collected (3 cows) or loss of the PRID (1 cow). Collection of Cervical Mucus Cervical mucus samples were collected from all cows 5 to 30 min before the single Al in the cows with normal estrus and before the first Al in the cows with induced estrus. Specifically, samples were collected after estrus signs were observed and before the end of estrus in cows with non induced estrus, 24 h after the removal of PRIDs, in Pq and Pq+ PGF2a-induced estrus and 48 h after the second injection of PGF2a. The vulva of the cow was washed with disinfectant solution, rinsed with water and then dried. Cervical mucus samples were collected using a plastic pipette 10 mL wide and 35 cm long (26). The front end of the pipette was cut at 0.5 cm length to make the opening wider and was smoothed to avoid wounding the genital tract. At the other end of the pipette, a 1m plastic, transparent tube was attached. Cervical mucus was collected from the external OS of the cervix and the area around it by means of oral aspiration. Cervical mucus was placed to a glass tube of 10 mL and transported to the laboratory in a refrigerated box (4“ C) and used within 2 hours after its collection. Aliquots of 1.5 to 2 mL of cervical mucus were homogenized in a Junk and Kunkel homogenizer and stored at -30’ C for future use in measurement of biochemical properties. Artificial Insemination After collecting the cervical mucus samples, all cows were inseminated with frozen semen of proven fertility, once the cows with normal estrus which presented estrus signs and twice the cows with induced estrus, 24 h and 48 h after the removal of PRlDs or 48 h and 72 h after the second dose of PGF2a. In the induced estrus groups, cows were not observed for estrus signs and double blind Al was performed. Spermatozoa were placed at the last third of the cervix for all cows. Pregnancy was determined by palpation per rectum 3 months after insemination. Biochemical Analysis Total protein. Total protein concentration in cervical mucus homogenate was determined by the method of Lowry et al. (8) as modified by Peterson (15). The same method was used by Prasad et al. (16). For this purpose a Sigma kitTUfor total protein measurement was used. The standard curve was done using total protein standard
44
Theriogenology
solution (400 vg purified bovine serum albumin /mL) and an aliquot of 0.1 mL homogenized cervical mucus were used. Results were expressed in pg/mL. Glucose. Glucose concentration was measured by the GOT-PERID method (25) using Boehringer reagents. Glucose standard solution (500mg/lOOmL) and an aliquot of 0.1 mL homogenized cervical mucus were used. Results were expressed in mg/lOO mL. Fructose. Fructose concentration was measured by the method of Mann (10) using Merk reagents. Fructose standard solution (100 ug/mL) and an aliquot of 0.2 mL homogenized cervical mucus were used. Results were expressed in pg/mL. Cholesterol. Cholesterol concentration was measured by the GOD-PAP method (20,21,25) using the kit of Elitech, that contains two reagents and a standard (Reagent 1: Pipes buffer, pH 6.90 SOmmollL, Phenol 24mmoVL and Sodium cholate O.Smmol/L. Reagent 2: Cholesterol esterase 2 200 U/L, Cholesterol oxidase 2 250 U/L, Peroxidase 2 1000 U/L and 4-Aminoantipyrine 0,5 mmol/L.)The same method used by Zaaijer et al. (29). Cholesterol standards in a range between 0.005 and 0.500 mmol/L and an aliquot of 0.1 mL homogenized cervical mucus were used. Results were expressed in mmol/L. Biochemical assays used in this study have been used by other researchers; accuracy, detection limits and quantification limits were acceptable. Statistical Analysis Analysis of variance (Anova) was used to compare cervical mucus biochemical properties among normal and induced-estrus groups. Comparisons between means were performed by Dunkan’s new multiple range test (22). Distribution was normalized by the proper transformation (logarithmic for total protein and fructose and reversal for glucose) where needed. The results were expressed as mean f SEM. A paired sample t-test was used to compare cervical mucus biochemical properties among the first three consecutive normal estrus cycles after delivery. T-test for independent samples was used to compare cervical mucus characteristics between normal estrus and the first estrus after the induced estrus. Chi square analysis was used to compare pregnancy rates among normal and induced-estrous groups and to estimate the percentages of cows in the induced-estrous groups that produced cervical mucus with similar physical properties to those of the normal estrous group. All analyses were performed using the statistical software program SPSS RESULTS Data showing biochemical properties of cervical mucus during three consecutive normal estrus periods after delivery are shown in Table 1. There were no differences in the biochemical properties of cervical mucus among the 15’, 2”d and 3’d estrus. Data showing biochemical properties of cervical mucus in the normal estrus after delivery (41 cows, 73 samples) and the first estrus after induction (20 cows, 20 samples) are shown in Table 2. There were no differences between these two groups. Therefore, all 61 cows constituted the normal estrus group.
45
Theriogenology
Table 1. Biochemical properties of cervical mucus (mean*SEM) in the first three consecutive normal estrus after delivery of 16 cows Cholesterol Fructose protein Glucose Total (mmol/L) (mg/lOO mL) (pg/mL) (pg/mL) Estrus 0.11 Ito. 5.2f0.7 3.6i2.2 1272.9*415.4 First 0.19*0.11 2167.4*1022.9 6.1*0.7 5.9*2.9 Second 0.11*0.05 17.2*10.9 1639.1*490.5 9.2*5.6 Third There were no statistically significant differences among groups. Table 2. Biochemical properties of cervical mucus (mean*SEM) in the normal estrus and in the first estrus after induction Estrus NED (41/73) FEI (20/20)*
Total protein (pg/mL) 1379.1 i153.7 1036.9*179.6
Glucose (mg/lOOmL) 7.7ti.2 4.9*0.9
Fructose (ug/mL) 4.6*1.4 5.9i4.1
NED: normal estrus after delivery FEI: first estrus after induction * Number of cows/number of samples used for the determination properties. There were no statistically significant differences between groups.
Cholesterol (mmol/L) 0.09*0.02 0.07&0.04
of biochemical
Data showing biochemical properties of cervical mucus in the normal (61 cows, 93 samples) and in the induced-estrus groups are shown in Table 3. The treatment used to induce estrus had an effect on total protein and cholesterol concentrations. Total protein concentrations and cholesterol concentrations were significantly lower in the normal-estrus group compared to those in the three induced-e&us groups (P c 0.001). Glucose and fructose concentrations were similar in the normal and in the inducedestrus groups. Table 3. Biochemical properties of cervical mucus (mean f SEM) in the normal and in the induced estrus groups Estrus group Normal (61193)’ Induced by Pq (18/18)* PGF2a (17/I 7)* P4+PGF2a (16/I 6)*
Total protein (ug/mL) 1330.2” f 134,5
Glucose Fructose (mg/l OOmL) (pg/mL) 7.3Of 1.9 4.ga* 1.3
Cholesterol (mmol/L) 0.09” f 0.02
6923.7b * 1631.5 6485.gb * 1740.7 5498.4b f 2006.8
16.O’f 3.2 8.3” * 1.7 11.7’ *3.2
0.87b f 0.25 0.65b * 0.21 0.86b f 0.40
12.3’ f 5.6 14.1’ * 5.6 6.1a *4.3
()* Number of cows/number of samples used for the determination of biochemical properties a,b’c Columns with different superscripts differ P
46
Theriogenology
Data showing pregnancy rates for normal and induced-estrus groups are shown in Figure 1. There were no differences in pregnancy rates among these groups, Finally, data showing percentages of cows in the induced-estrus groups with similar biochemical properties (mean&EM) to that in the normal estrus are shown in Table 4. Only a small percentage of cows in induced-estrus groups produced at the day of first Al cervical mucus had biochemical properties similar to those in the normalesJus group.
0 60
0
Normal Induced by
Normal
m
p4
m
PGFza
m
Pb+PGFza
\
estrus
I Induced estrus
Figure 1. Pregnancy rates in the normal and in the induced estrus groups. There were no statistically significant differences among groups. Table 4. Percentages of cows with similar biochemical properties (mean*SEM) in normal and induced-estrus groups Normal Biochemical properties
p4
meankSEM Total protein II 330.2k134.5
Wmb
5.9
n=18 mean&SE F232.6)
30.8
(758.5k243.20.0
(770.2k336.3)
5)
Glucose < 7.3zt1.9 (mgll OOmL) Fructose I4.9k1.3
(ug/mL) Cholesterol
%
Estrus induced by PGF2a P4+PGF2a n=16 n=17 % mean&EM Oh mean&EM
I 0.09*0.02
50.0
(5.6k0.9)
70.6
(4.5k0.7)
66.7 (5.820.8)
70.6
(I. 1kO.6)
66.7
(0.9kO.5)
84.6 (0.4*0.4)
22.2
(0.05*0.02)35.3
(0.06*0.01)
60.0 (0.04&0.01)
Theriogenology
DISCUSSION In the present study, it has been found that biochemical properties of cervical mucus collected in the first three consecutive normal estrus cycles after delivery do not differ significantly. Biochemical properties of cervical mucus collected during the first estrus after the induced one also do not differ from those of normal estrus. Therefore, the first estrus after the induced could be considered a normal one in terms of biochemical properties. However, only total protein and cholesterol concentrations differ in the normal and in the induced-estrus groups. On the contrary, glucose and fructose concentrations did not differ. Additionally, the percentages of cows in the induced-estrus groups producing at the time of the first Al cervical mucus with total protein and cholesterol concentrations similar to those in the normal-estrus group, were very low. Furthermore, cervical mucus collected from non induced estrus was abundant, clear and transparent, while that from most induced-estrus cows was thicker and less abundant. Total protein concentration in the cervical mucus collected from cows with PGF2ainduced estrus was similar to that observed by Prasad et al. (16). Furthermore, Prasad et al. (16) found that total protein concentration is higher (almost double) in cervical mucus collected the day before estrus manifestation than that collected during the day of estrus in the PGF2a-induced estrus group. They observed lower total protein concentrations on the day of estrus. They also observed increased proteins in cervical mucus collected from cows that did not exhibit estrus signs, as well as in cervical mucus collected from controls during Day 12 of the estrous cycle (16). In the present study, total protein concentration in the PGF2a-induced estrus group was similar to that in normal estrus for only 30.8W of cows at the day of first Al. Total protein concentration could be reduced when the second Al was performed. However, this suggestion must be studied further. Additionally, Rexroad and Barb (17) observed that total protein concentration was higher in the cervical mucus of ewes when estrus was induced by progesterone-impregnated sponges than it was in the control group, but it was lower in cervical mucus of ewes that had received MAP than it was in the control group. Cholesterol concentration in the cervical mucus of cows with normal estrus is similar to that observed by Zaaijer et al. (29). Contrary to Zaaijer et al. (29) who found lower cholesterol concentration in cervical mucus collected from cows with progesterone (PRID) and prostaglandin F2a-induced estrus compared to the results of the present work. Additionally, Zaaijer et al. (29) found similar cholesterol concentration in cows with normal and with induced estrus, but estrus was exhibited 72 h after PRID removal, that is, when a second Al was performed in the present study. It is supposed that in the present study cholesterol concentration might be reduced during the second Al. In the present work, 60% of cows with P4+ PGF2a-induced estrus had cholesterol concentrations similar to those of cows with normal estrus, and the mean value is similar to that reported by Zaaijer et al. (29). Glucose and fructose concentrations of cervical mucus do not differ significantly in cows with normal and induced estrus. On the contrary, El-Naggar and Horrath (3) found higher fructose concentration in the cervical mucus of cows with normal estrus than that observed in the present study, but the method they used for determination of fructose was criticized by Mann (11). Furthermore, the fructose concentration found in the
48
Theriogenology
present study cannot be compared to that found in other studies (27) because of the different determination methods used. Glucose concentrations in our study were lower than those reported by Weed and Carrera (28) in the cervical mucus of women. On the contrary, Bane and Rajakoski (1) merely traced glucose in the estrous cervical mucus of cow, using calorimetric tips, while Zaaijer and Van der Host (30) observed differences in glucose concentration depending on ovulation and conception. Furthermore, glucose and fructose concentrations found in the present study were significantly decreased in a high percentage of cows with induced estrus on the day of first Al. The reason for that must be investigated. Despite the different total protein and cholesterol concentrations of cervical mucus among normal and induced estrus groups, pregnancy rates did not differ significantly among these groups. Therefore, biochemical properties of cervical mucus do not directly affect fertility. In the induced-oestrus groups a second Al was performed 24 h after the first one. Biochemical properties of cervical mucus could be similar to those in normal estrus at the time of second Al. Furthermore, the percentages of cows in the induced-estrus groups producing at the time of the first Al have similar cervical mucus as in the normal estrus are very low. The biochemical properties that mostly differ are the total protein and the cholesterol concentrations. It has been found that cows do not exhibit estrus at a determined time (6,18). In summary, the results from the present study showed that biochemical properties of cervical mucus are similar during the first three consecutive normal estrus periods after delivery; that the first estrus after an induced one can be considered as a normal estrus; that total protein and cholesterol concentrations are significantly different in the normal and in the induced-estrus groups; that a small percentage of cows with induced estrus produces at the day of first Al cervical mucus with concentrations of total protein and cholesterol similar to those for cows with normal estrus. These two properties could be used as indicators of the optimal time for Al after induced estrus. REFERENCES 1.
2. 3.
4. 5. 6.
Bane A, Rajakoski E. The bovine estrus cycle. Cornell Vet 1961;51:77-95. Bergman P. Sexual cycle, time of ovulation, and time of optimal fertility in women. Acta Obst Gynec Scandinav 1960;29(Suppl. IV):6-139. El-Naggar MA, Hornath EB. Sugar content of the cervico-vaginal mucus of cattle during the sexual cycle with special reference to fructose. Acta Vet Hung 1971;21:15-20. Elstein M. The cervix and its mucus. In Clinics in Obstetrics and Gynecology. Vol 1, No 2, 1974: 345-368. Haynes NB, Lamming GE. The carbohydrate content of sow uterine flushings. J Reprod Fertil 1967;14:335-337. Garcia-Winder MJ, Gallegos-Sanchez J. Estrus synchronization in Holstein cows using reduced doses of prostaglandin Fza. Theriogenology 1991;36: 191199.
Theriogenology
7. 8. 9. 10.
11.
12. 13. 14. 15. 16.
17. 18. 19. 20. 21. 22. 23.
24. 25. 26.
27.
28. 29.
30.
49
Gibbons RA. Chemical properties of two mucoids from bovine cervical mucin. Biochem J 1959;73:209-225. Lowry CH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin Phenol reagent. J Biol Chem 1951;193: 265275. Lutwak-Mann C. Some properties of uterine and cervical fluid in the rabbit. Biochim Biophys Acta 196258: 637-639. Mann T. Studies on the metabolism of semen. 3. Fructose as a normal constituent of seminal plasma. Site of formation and function of fructose in semen. Biochem J 1946;40:481. Mann T. Energy requirements of spermatozoa and the cervical environment. In The Biology of the Cervix. Eds. R J Blandau and KS Moghissi. University of Chicago Press. 1973:334. Marcus SL. The cervical factor in infertility. J Reprod Med 1969;3:138-142. Moghissi KS. The function of the cervix in fertility. Fertil Steril 1972;23:295-306. Odeblad E. The functional structure of human cervical mucus. Acta Obstet Gynecol Stand 1968;47 (Suppl. 1):57-64. Peterson GL. A simplification of the protein assay method of Lowry et al. Which is more generally applicable. Anal Biochem 1977;83:346-356. Prasad A, Kalalyan NR, Bachlaus NK, Arora RC, Pandey RS. Biochemical changes in the cervical mucus of buffalo after induction of oestrus with prostaglandin FZaand cloprostenol. J Reprod Fertil 1981;62:583-587. Rexroad CE, Barb CR. Cervical mucus in estrous ewes after treatment with estrogen, progestogens and intrauterine devices. J Anim Sci 1977;44: 102-l 05. Roche JF, Ireland J, Mawhinney S. Control and induction of ovulation in cattle. J Reprod Fertil 1981;30 (Suppl): 21 l-222. Schumacher GFB. Biochemistry of cervical mucus. Fertil Steril 1970;21:697705. Siedel J, et al. J Clin Chem Clin Biochem 1981;19:838. Stahler F, et al. Med Lab 1977;30:29. Steel RGD, Torrie JM. Principles and Procedures in Statistics. New York McGraw-Hill. 1980 Suga T, Masaki J. Studies on the uterine secretion of the cow. 6. Sugar and polyol constituents in the luminal fluid of bovine uterus. Jap J Anim Reprod 1973; 18: 143-I 47. Trakatellis A. Cholesterol metabolism. Biochemistry. 1986:559-576. Trinder P. Ann Clin Biochem 1969;6:24. Tsiligianni Th, Karagiannidis A, Brikas P, Saratsis Ph. Relationship between certain physical properties of cervical mucus and fertility in cows. Dtsch Tierarztl Wochenschr 2000; 107 (1):28-31. Van der Linden PJQ, Kets M, Gimpel JA, Wiegerinck MAHM. Cyclic changes in the concentration of glucose and fructose in human cervical mucus. Fettil Steril 1992;57: 573-577. Weed JC, Carrera AE. Glucose content of cervical mucus. Fertil Steril 1970;21:866-872. Zaaijer D, Counotte GHM, Sol J, Smidt WJ, Broadbent PJ. Changes in the composition of cervical mucus of the cow during the estrous cycle as parameters for predicting potential fertility. Theriogenology 1993;39:569-580. Zaaijer D, Van der Horst CJG. Cyclical changes in hormones, carbohydrates and indole metabolism in cervical mucus of normal, fertilizing cows and the relationship with non-fertility. Cytobios 1983;37: 113-I 27.
50
31.
Theriogenology
Zavy MT, Clark WR, Sharp DC, Robberts RM, Bazer FW. Comparizon of glucose, fructose, ascorbic acid and glucose phosphate isomerase enzymatic activity in uterine flushings from non-pregnant and pregnant gilts and pony mares. Biol Reprod 1982;27:1147-1158.