Non-fertility in cows: treatment with PGF and investigation of uterine biopsies

Experiments with seventeen 'repeat breeders' confirmed that the carbohydrate composition, the hormonal pattern, the cholesterol content and the occurrence of indole metabolites, such as blue fluorescing protein, in cervical mucus can be used as parameters for the fertilizing capacity of these cows. After careful rectal examination 25 mg PGF2 alpha was administered to thirteen 'repeat breeders' on that day of the luteal phase when next to the corpus luteum no follicle was palpable; ten of them became pregnant or were fertilized. Uterine biopsies were taken from eight 'normal' cows and from eight 'repeat breeders', simultaneously with cervical mucus samples. The biochemical composition of the biopsies was comparable to that of the mucus samples. The taking of two biopsies produced injurious consequences for the animals, perceptible in oestrous behaviour. Investigation of cervical mucus samples is therefore preferred to uterine biopsies for the examination of sterility in cows.     

Carbohydrate metabolism and the temporal occurrence of a serotonin-like indole in the male and female reproductive tract and its relationship with PGF and infertility: a review

Both in the male and in the female reproductive tract glucose can be converted via either the pentose pathway or the sorbitol pathway. It is shown that a disturbed carbohydrate metabolism can lead to infertility, i.e. in the cow and in the bull semen. Evidence is provided that the serotonin-like indole which occurs in the protein-complex and is liberated, can exert effects on the uterine endometrium comparable to those caused by serotonin. It is suggested that the indole liberated can cause ischaemia, followed by regression of the endometrium. When this occurs in repeat breeder cows, exogenous PGF given in mid-cycle on a suitable day, may restore the endometrium so that the cow can again become pregnant. The possibility is mentioned that in humans the free indole might cause regression of the endometrium and some distress symptoms, but thereafter endogenous PGF does increase the vascular permeability resulting finally in bleeding.     

Physical properties of bovine cervical mucus during normal and induced (progesterone and/or PGF2alpha) estrus

Ninety two Friesian cows were used to determine physical properties of cervical mucus collected during normal estrus and estrus induced. Estrus was induced using either progesterone (P4) releasing intravaginal devices (PRID) and/or prostaglandin F2alpha (PGF2alpha). The animals were assigned to 4 groups (no treatment, a PRID for 12 days plus an injection of 1000 IU PMSG at the removal of the PRID, a double injection of 3 mL PGF2alpha 11 days apart, and a PRID for 7 days plus an injection of PGF2alpha 24 h before the removal of PRID). A number of cows with normal estrus exhibited three consecutive estrus cycles after calving. Cows that had not shown estrus for three months after calving had their reproductive system palpated twice at 10-day intervals, to determine their ovarian activity. Then PRID and/or PGF2alpha was administered to cows that were found to have a palpable corpus luteum in one of two palpations (cycling cows). The cows of the three induced estrous groups were artificially inseminated (AI) twice, while those with normal estrus received only a single AI. Cervical mucus samples were collected from all cows 5 to 30 min before the first AI. Additionally, samples of cervical mucus were collected from 20 cows at their first estrus after the induced estrus. The results are summarized as follows: 1) The physical properties of cervical mucus were similar in the first three normal consecutive estrus cycles after calving. 2) The physical properties of cervical mucus in normal estrus after calving were similar to those in the first estrus after an induced estrus. 3) The pH values for normal estrus were similar to those for induced estrus. 4) Viscosity of cervical mucus in the normal estrous group was significantly lower than that in the induced estrus. Furthermore, significant differences were noticed among the three induced estrous groups. 5) Spinnbarkeit, crystallization and receptivity of cervical mucus (penetration test) were significantly higher in the normal estrous group than in the induced estrous groups, while no difference was detected among induced estrus groups. 6) Pregnancy rates in the normal estrus group were the same as in the induced estrus groups. 7) The percentages of cows in the induced estrous groups that produced cervical mucus with similar viscosity, spinnbarkeit and receptivity (penetration test) characteristics as the normal estrus group, was very low.     

Chemical properties of bovine cervical mucus during normal estrus and estrus induced by progesterone and/or PGF2alpha

Ninety-two Friesian cows were used to determine the chemical properties of cervical mucus during normal estrus and estrus induced by progesterone (P4)-releasing intravaginal devices (PRID) and/or prostaglandin F2alpha. 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 i.m. injection of PGF2alpha 11 days apart, or PRID for 7 days plus an im injection of PGF2alpha 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 10-day intervals, to determine their ovarian activity. Then PRID and/or PGF2alpha was administered in cows that had a palpable corpus luteum in one of the two palpations (cyclic cows). A double artificial insemination (AI) was performed to the cows of the three induced-estrus groups, while the cows with normal estrus received only one AI. Cervical mucus samples were collected from all cows 5 to 30 min before the first AI. 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 < 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.     

Cyclic changes in hormones, carbohydrates and indole metabolism in cervical mucus of normal, fertilizing cows and the relationship with non-fertility

In the cervical mucus of 'normal' cows the cholesterol content was very low at D--0; at D + 12 it was about fifty times as high; then it decreased to about half its value at D + 17 and then to the low value at D--0. Usually small amounts of oestrogen, testosterone, pregnanedione and progesterone were found at D--0; on D + 12 more pregnanedione was found and less oestrogen, while at D + 17 more oestrogen occurred and less pregnanedione. The fructose and glucose content was very low at D--0; then it increased until D + 12, when glucose was dominant, while at D + 17 it had decreased and rather large amounts of glucuronic acid and of sorbitol occurred. On D + 12 a blue fluorescing indole metabolite was sometimes found. Deviations from these patterns, were found particularly in the winter months, and coincided with lowered fertility. Indole metabolism was stronger in the winter months than in the summer months and occurred more in cows than in heifers.     

Antibodies to egg yolk in blood serum of rabbits and cattle and cervical mucus of cattle inseminated artificially.

Serum titers to egg yolk were induced in 6 rabbits by intravaginal deposition of an egg-yolk citrate extender used for artificial insemination of cattle. There was no effect of the low serum titers to egg yolk on fertility of the inseminated rabbits. Titers to egg-yolk semen extender were found in 3% of 59 cows of normal fertility compared to 29% of 14 repeat breeder cows of low fertility, all previously inseminated with semen diluted with egg yolk-citrate extender. Four of 6 cervical mucus samples (67%) from the repeat breeder cows had high titers to egg yolk, but only one also had a positive titer in blood serum.     

Culture of uterine flushings, cervical mucus, and udder secretions collected post-abortion from heifers artificially exposed to Brucella abortus

Uterine flushings, cervical mucus swabs and udder secretions collected at weekly intervals from five mixed breed beef cows (four Brucella abortus strain 19 vaccinates, and 1 non-vaccinate) were cultured for Brucella abortus . Prior to sampling, four of the five had aborted 7-to 8-month-old fetuses and one gave brith to a weak calf. The fetuses and/or udder secretions from the cows were culture positive for B. abortus at the time of parturition. Three of the cows developed persistent udder infections. Two of these cows were also shown to have brucellae in their cervical mucus for 10 and 20 days and in their uterine flushings for 17 and 41 days after parturition, respectively. One other cow had brucellae in the cervical mucus for 16 days and in the uterine flushings for up to 36 days post-abortion. All attempts to isolate the organism from this cow's udder secretions in culture were negative. In two cows with culture-positive uterine flushings, isolations of brucellae were made subsequent to normal postpabortion return to estrus.     

Changes in the composition of cervical mucus of the cow during the estrous cycle as parameters for predicting potential fertility

Four experiments were conducted to test the hypothesis that the composition of cervical mucus can be used as an indicator of reproductive efficiency in the cow. In Experiment 1, biochemical changes were studied in cervical mucus during the estrous cycle. Sorbitol concentration was observed to be highest at 1 to 3 days prior to estrus and lowest on Days 6 to 12 (P<0.001) of the estrous cycle. Cholesterol and protein concentrations were highest at Day 6 of the estrous cycle and lowest on the day of estrus (P<0.001). In Experiment 2, the relationships between the biochemical characteristics of cervical mucus and fertility were studied. It was shown that the embryo transfer recipients which exhibited a high concentration of sorbitol (>1.5 mMol/l) at 1 to 3 days before estrus; a low concentration of protein (< 2 units); and a low concentration of cholesterol (<0.1 mMol/l) on the day of estrus had a higher level of fertility than their counterparts. The predictive ability of these criteria was tested using embryo transfer recipients (n=294) in Experiment 3. Significantly more of the animals predicted to have high potential fertility became pregnant than those predicted to have low potential fertility (70.7 vs 45.6%; P<0.001). A similar difference in pregnancy rate for cows (n=56) presented for artificial insemination was observed in Experiment 4 (59.1 vs 27.2%; P<0.10). These results suggest that the composition of cervical mucus may be a useful indication of potential fertility in cattle.     

Cytologic atypias in the uterine fluid of intrauterine contraceptive device users

A peculiar cell pattern, characterized as an atypical glandular cell cluster (AGCC), was found in the uterine fluid from women using intrauterine contraceptive devices (IUDs). This atypia can mimic adenocarcinoma. Since the atypical cells were present in much higher frequency in the uterine fluid than in the cervical mucus, it is suggested that they originated in the endometrium.     

Composition and function of human cervical mucus

Human cervical mucus was collected from seven donors during the follicular, ovulatory and luteal phases of the ovulatory menstrual cycle. Individual mucus samples were solubilized and fractionated on Sepharose columns into excluded mucins and low-molecular-weight proteins. Mucin fractions were highly purified, as evidenced by the presence of a single N-terminal amino acid residue, threonine, and by the absence of contaminating plasma proteins. Amino acid compositions of mucins isolated during different menstrual phases of a single donor or from different donors were similar. Mucin carbohydrate compositions were also similar, except for the sialic acid-to-fucose ratio, which varied significantly between donors but not within the menstrual cycle of a single donor. An analysis of variance was applied to evaluate the contribution of mucin composition to viscoelasticity, as quantitated by microrheometry. Viscoelasticity was dependent on the donor, on the percent nondialyzable solids and on the mucin content, but not on the phase of the menstrual cycle during which the sample was collected. These findings suggest that mucus function (viscoelasticity) is reflected in carbohydrate composition and/or structure and that this menstrual relationship is unique for each donor. Furthermore, the absence of menstrual phase-dependent differences in mucins suggests that mucin concentration and not composition changes in response to alterations in the hormonal milieu.     

Impact of estrous synchronization methods on cellular proportions in cervical mucus and serum hormone concentrations

The objective of this study was to examine cytological changes of cervical mucus following the induction of estrus with intra-vaginal drug release (CIDR) devices in dairy cows. Sixty healthy Holstein Frisian cows, averaging 80 (+/-10) days post-partum, were selected from a commercial dairy farm around Shiraz. Cows in the control group were synchronized by the Ovsynch protocol. Cows in the second group (OV+CIDR) were subjected to the same Ovsynch protocol but in addition were administered a progesterone-releasing CIDR. Cows in the third group (OV+S-CIDR) were subjected to Ovsynch procedures but received the skeleton of a CIDR device, which did not release progesterone. Cows in the fourth group (E2+CIDR) received a progesterone releasing CIDR but were injected with estradiol benzoate. Cows in group 5 (E2+S-CIDR) received a CIDR skeleton and estrodial benzoate. CIDR devices were removed from cows in groups 2-4 and all cows were injected with PGF2alpha on day -3. Blood samples and cervical mucus discharges were collected from all cows on days -10, -3, 0 and 12 relative to AI. On the day of AI, the mean+/-S.D. percentage of neutrophils was significantly higher (p<0.05) in the S-CIDR+OV and S-CIDR+E2 groups than in Ovsynch group. Comparing the percentage of neutrophils in cervical mucus of Ovsynch group (less than 1%) with that of other treatment groups on the day of AI (from 5 to 39%) revealed the influential effect of a CIDR device on the reproductive tract. Results of the current study did not reveal hormonal effects but did identify mechanical effects of CIDRs on cell percentages in cervical mucus. The hormonal effects were probably masked by mechanical effects. Therefore, we were not able to confirm hormonal effects on proportions of different cells in cervical mucus. Consequently, additional research on hormonal effects and the mechanical effects of CIDR on the uterus is required.     

Studies on bonnet monkey cervical mucus the effect of proteases on mucus glycoproteins of macaca radiata

The influence of proteinases on monkey cervical glycoproteins was investigated to assess their effect on cervical mucus and, thereby, on sperm penetration. The major component of periovulatory cervical mucus, a high molecular weight glycoprotein, was treated with Pronase, trypsin, chymotrypsin, papain, and bovine seminal peptidase, and the enzyme-resistant glycoprotein was purified by gel filtration on Sepharose 2B. A macromolecular component in high yield was recovered containing carbohydrate and protein moieties. Asialoglycoprotein, on treatment with Pronase, trypsin, and bovine seminal peptidase released more than one glycoprotein fragment. The carbohydrate and amino acid components of the native and degraded glycoproteins were similar in composition with variations in proportions. The structure of the carbohydrate-rich, pronase-resistant glycoprotein, further purified on Sepharose 2B, was examined. Sequential Smith degradation and methylation of the degraded glycoprotein fragment established a structure that shows some differences to that of the native glycoprotein. The influence of proteinases on cervical-mucus glycoproteins and a possible mechanism of sperm penetration through Pronase-treated glycoproteins is discussed.     

Lipopolysaccharide (LPS) suppresses follicle development marker expression and enhances cytokine expressions,which results in fail to granulosa cell proliferation in developing follicle in cows

Postpartum endometritis is known to be associated with ovarian dysfunction in cows. Lipopolysaccharide (LPS) generated by Gram-negative bacteria is recognized by toll-like receptor 4 (TLR4), which leads to an inflammatory response by the generation of cytokines such as tumor necrosis factor-α (TNF-α) and interleukins. In this study, we investigated the effect of endometrial LPS on granulosa cell functions during early follicular development in cows. Uteri and follicles were obtained from a slaughterhouse and classified into either clinical endometritis (CE) or normal groups by vaginal mucus test. TLR4 mRNA and protein in normal cows were expressed in granulosa cells collected from follicles measuring 1–3 and 4–7 mm in a diameter, respectively. LPS content in endometrium and follicular fluid of CE cows was significantly higher than that in normal cows. Compared to normal cows, CE cows showed lower expression of follicular development markers (FSHR, CYP19A1, CCND2, and LHCGR) in granulosa cells, lower estradiol-17β concentrations in follicular fluid, and lower granulosa cell proliferation. CE contraction significantly increased cytokine expressions (TNF, IL-1A, and IL-1B) in granulosa cells and suppressed apoptosis of granulosa cells compared to normal cows. LPS significantly suppressed the expression of follicular development markers and the production of estradiol-17β in granulosa cells and reduced granulosa cells proliferation compared to cells cultured without LPS. LPS significantly increased cytokine expressions and suppressed granulosa cell apoptosis. Thus, the present results suggest that the existence of LPS in developing follicles is one of the causes of ovarian quiescence in cows.     

Impact of methodological factors on sperm penetration into cervical mucus in cattle

The aim of this study was to investigate the influence of methodological factors on the interaction of bovine spermatozoa and homologous cervical mucus. Cervical mucus was obtained from three cows during estrus. To evaluate the penetration ability of frozen-thawed semen samples of five different bulls, fresh mucus as well as frozen-thawed mucus, stored for 1, 10 or 30 d in liquid nitrogen, were used. Penetration assays were performed at 38 degrees C for 10 min, and the most advanced spermatozoon was located and the distance determined. Semen parameters were examined by a computer-assisted videomicrographic system. Conservation of mucus in liquid nitrogen for up to 30 d did not influence the results of the penetration assay. In contrast, the mucus of individual cows showed significant differences in the migration distance of spermatozoa. Sperm concentration, mean velocity and number of forward moving spermatozoa were significantly correlated with mucus penetration. These results demonstrated that the mucus penetration assay in cattle can be performed by dividing a mucus sample from a cow into many portions and storing the sample in liquid nitrogen.     

Role of tissue kallikrein-related peptidases in cervical mucus remodeling and host defense

Human tissue kallikrein-related peptidases (KLKs) are 15 hormonally regulated genes on chromosome 19q13.4 encoding secreted serine proteases. Many KLKs are expressed throughout the female reproductive system and found in cervico-vaginal fluid (CVF). Immunohistochemistry was performed to determine KLK localization in the female reproductive system (fallopian tube, endometrium, cervix and vagina tissues). KLK levels were measured in CVF and saliva over the menstrual cycle to study whether KLKs are regulated by hormonal changes during the cycle. In vitro cleavage analysis was performed to establish whether KLKs may play a role in vaginal epithelial desquamation, mucus remodeling or processing of antimicrobial proteins. KLKs were localized in the glandular epithelium of the fallopian tubes and endometrium, the cervical mucus-secreting epithelium and vaginal stratified squamous epithelium. KLK levels peaked in CVF and saliva after ovulation. In vitro cleavage analysis confirmed KLKs 5 and 12 as capable of digesting desmoglein and desmocollin adhesion proteins and cervical mucin proteins 4 and 5B. KLK5 can digest defensin-1alpha, suggesting it may aid in cervico-vaginal host defense. We provide evidence of potential physiological roles for KLKs in cervico-vaginal physiology: in desquamation of vaginal epithelial cells, remodeling of cervical mucus and processing of antimicrobial proteins.     

Biochemical changes in cervical mucus-factor infertility

Cervical-factor infertility has generally been attributed to the presence of antispermatozoal antibodies in the secretions of the uterine cervix, despite the fact that the incidence of sperm-specific antibodies in these women is generally low. We report here a modification in the structure of the cervical mucus of patients with a diagnosed cervical factor. Cervical mucus from patients with a cervical factor of nonimmunological origin, collected during the periovulatory phase of the menstrual cycle, had (1) a significant decrease in the content of glycosidically bound sialic acid and (2) an increased ability to act as an acceptor for sialic acid from cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-sialic acid) when incubated with an exogenous sialyltransferase; in comparison to mucus from normal fertile women. Both siaiyltransferase and fucosyltransferase activities were detected in cervical mucus, but there was no difference between fertile normal and cervical-factor patients using the assays described. These results reinforce a possible role for sialic acid residues and their associated glycosyltransferases in the regulation of spermatozoal–cervical mucus interaction.     

Comparative study on mucus glycoproteins in rat stomach and duodenum

The density of mucus glycoprotein compared to that of the corpus, antrum and duodenum was; 1.52, 1.49 and 1.57 g/ml respectively. Carbohydrate composition of gastrointestinal mucus glycoprotein consisted of N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and sialic acid. Ratios of carbohydrate composition among corpus, antral and duodenal mucus glycoproteins differed. The average length of an oligosaccharide was found to be about 12-13, 14 and 10 sugars in the corpus, antrum and duodenum, respectively. In the corpus, the amino acid content was found to have the following quantitative order: Thr greater than Ser greater than Glx = Pro; in the antrum: Thr greater than Ser greater than Glx; and in the duodenum: Thr greater than Ser greater than Pro. Corpus, antral and duodenal mucus glycoproteins have the blood-group A antigen; antral mucus glycoprotein in particular exhibited strong blood-group A activity.     

Sex steroids in cervical mucus of spontaneous or induced ovulatory cycles

Evaluation of sex steroids in cervical mucus was performed at different phases of spontaneous or clomiphene-citrate-induced ovulatory cycles. To this end, 11 women with normal ovulatory cycles and 9 subjects with polycystic ovary syndrome of comparable age and body mass index were investigated. Serum and cervical mucus samplings were assessed for 17beta-estradiol (E2), progesterone, testosterone, and sex hormone binding globulin levels at the pre-, peri-ovulatory, and mid-luteal phases of the cycle. The cervical mucus maturation index also was estimated in all women. Measurable amounts of E2 were found in most mucus samples with a cyclic variation in all cases. The highest E2 and mucus maturation index values coincided, but both lagged by 24 h behind the serum mid-cycle peak of this steroid. Detectable amounts of progesterone were found in the luteal phase, testosterone was present at low levels throughout the cycle, but sex hormone binding globulin was undetectable in all cervical mucus samples. Differences between spontaneous or drug-induced ovulatory cycles were not found. It is concluded that sex steroids are present in human cervical mucus, showing variations similar to those in peripheral blood. The significance of these findings is not clear at present, but it is probably related to the cyclic changes of cervical epithelium and gland secretion. An important implication of the absence of measurable sex hormone binding globulin amounts in cervical mucus is that the free fraction of sex steroids present in that fluid are presumably higher, and therefore, expected to exert greater biologic activity than in peripheral blood.     

Biochemical differences between trail mucus and adhesive mucus from marsh periwinkle snails

The composition of the adhesive form of marsh periwinkle mucus was compared to the trail mucus used during locomotion. The trail mucus consists primarily of large, carbohydrate-rich molecules with some relatively small proteins. In contrast, the adhesive mucus has 2.7 times as much protein with no significant difference in carbohydrate concentration. The resulting gel has roughly equal amounts of protein and carbohydrate. This substantial increase in protein content is due to the additional presence of two proteins with molecular weights of 41 and 36 kD. These two proteins are absent from the trail mucus. Both proteins are glycosylated, have similar amino acid compositions, and have isoelectric points of 4.75. This change in composition corresponds to an order of magnitude increase in tenacity with little clear change in overall concentration. The difference between adhesive and non-adhesive mucus suggests that relatively small proteins are important for controlling the mechanics of periwinkle mucus.     

Role of sialic acid and sulfate groups in cervical mucus physiological functions: study of Macaca radiata glycoproteins

The influence of charged groups in glycoproteins was investigated to assess their effect on the physiological functions of bonnet monkey cervical mucus. The macromolecular glycoproteins from peri-ovulatory, midcycle phase cervical mucus were treated with Pronase, trypsin and chymotrypsin and the enzyme-resistant glycoproteins purified by gel filtration on Sepharose 4B and a high molecular weight component containing carbohydrates, proteins and sulfate groups was recovered in high yield. This material still reacted with an antiserum directed against purified midcycle glycoprotein but not against another antiserum directed against luteal phase purified glycoproteins. Upon treatment with Pronase, trypsin and chymotrypsin, asialoglycoproteins and desulfated asialoglycoproteins released fragments of low molecular sizes, none of which reacted with the anti-midcycle glycoprotein antiserum. Cervical mucus collected from the estrogenic phase displayed a morphology supporting sperm migration, and this mucus retains the same morphology and reacts with the anti-midcycle glycoprotein antiserum following mild treatment with sialidase and subsequently with Pronase. These results imply that charged carbohydrate groups help maintain the structural and functional integrity of the mucus glycoprotein in its biological environment.