Vaginal fluid ferning cytology-Crystallization of Bovine Cervical Mucus at Oestrus: An Update

Bitches exhibited a characteristic arborization pattern of the fluid from the anterior vagina during pro-oestrus and oestrus. These changes were monitored together with conventional vaginal cytology and plasma oestrogen and progestagen concentrations. A classical ferning pattern, similar to that seen in bovine cervical mucus at oestrus, occurred after the peak in plasma oestrogen concentrations. Ferning was most intense after the second peak of cornification of vaginal epithelial cells. It is suggested that a 'Ferning Index', when combined with conventional vaginal cytology, can be of use in determining the optimum mating time in the bitch.

Eve spanked elasticity of cervical-vaginal secretions is abnormal in polycystic ovary syndrome: Case report of five PCOS women. Secretions able to crystallize are characterized by containing mucoproteins e. The central axis is short, with or without venations and subvenations, which are irregular 1 Figure 2C. Obstet Gynecol. Undersokelse av cervikalslim for diagnose av tidlig drektighet og endokrint betingede forstyrrelser av seksualcyklus hos husdyr. Type B crystallization corresponds to that observed when mucus is semi-translucent, Vaginal fluid ferning cytology and easily obtained from the cervix.

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Slowly release the pressure exerted on the bulb. In such cases, prolonged lack of receptivity may suggest the need to artificially Vaginal fluid ferning cytology or force-breed the animal. View several fields to get an overall visual idea of Vaginal fluid ferning cytology percentage of cornified cells. As a canine fertility test, they are very useful for timing:. Superficial cells Vxginal a pyknotic nucleus and angular cytoplasm. National Center for Biotechnology InformationU. The peak in follicle-stimulating hormone levels signals ovulation and entry into estrus 10, Other methods can provide additional details. The cytoplasm may even become angular. Click here to view larger figure. Anestrus Intermediate and parabasal cells predominate in smears taken during anestrus. Non-cornified Parabasal cells have a large stippled nucleus and a rounded cytoplasm The nucleus is large compared to the cytoplasm. Entry into metestrus coincides with a continuous rise in progesterone hormone levels 6 Lesbien stickers corresponds to the beginning of human luteal phase 12 Figure cytollgyMetestrus, left panel. Determining the fertile period fdrning breeding fenring is the biggest challenge dog breeders face in consistently achieving all the big litters they want.

PIP: The effectiveness of intrauterine instillation of solutions for midtrimester abortions is well established, but the mechanism for triggering the onset of labor is unclear and represents a challenging problem for obstetricians and physiologists.

  • Stages of the canine estrous cycle can be defined by sexual behavior, physical signs vulvar swelling, vaginal bleeding or by vaginal cytology.
  • Superficial cells have a pyknotic nucleus and angular cytoplasm.
  • Having a canine fertility test you can use at home is invaluable.
  • Fern test refers to detection of a characteristic ' fern like' pattern of vaginal secretions when a specimen is allowed to dry on a glass slide and is viewed under a low-power microscope.
  • A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration or regeneration following pathological challenge.

Fern test refers to detection of a characteristic ' fern like' pattern of vaginal secretions when a specimen is allowed to dry on a glass slide and is viewed under a low-power microscope. It also may provide indirect evidence of ovulation and fertility, although it does not predict the time of ovulation. Ferning occurs due to the presence of sodium chloride in mucus under estrogen effect. When high levels of estrogen are present, just before ovulation, the cervical mucus forms fern-like patterns due to crystallization of sodium chloride on mucus fibers.

This pattern is known as arborization or 'ferning'. When progesterone is the dominant hormone, as it would be in the second half of a normal cycle, the fern pattern is no longer discernible, and the pattern is completely absent by the 22nd day of a woman's cycle. The disappearance of the fern pattern after the 22nd day suggests ovulation, and its persistence throughout the menstrual cycle suggests an-ovulation infertility.

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The onset of diestrus also correlates well with loss of fertility, and breedings after the diestrus shift are rarely fertile. Induction of overt menstruation in intact mice. The murine reproductive or estrous cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. These changes in cell typology are indicative of underlying endocrine events. Intermediate and parabasal cells predominate in smears taken during anestrus.

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Manuel E. Rev Med Vet. Bovine cervical mucus changes its biochemical composition and biophysical properties due to the variations in sex steroid levels during the oestrous cycle. The objective of this article is to provide an updated review of the main aspects regarding crystallization of bovine cervical mucus.

First, it makes reference to the composition of cervical mucus and the critical functions that this secretion exerts on bovine reproductive physiology, as well as in other species. Then, the article deals with the phenomenon of crystallization observed in cervical mucus, describing the main models used to classify the crystalline patterns observable in mucus at oestrus stage some of them resembling ferns, palm leaves and stellar patterns, among others.

Finally, it addresses the importance of the phenomenon of cervical mucus crystallization for the understanding of bovine reproductive physiology. Keywords: Bovine physiology, cervical mucus, crystalline pattern, crystallization, oestrus. From the onset of cow domestication thousands of years ago, man has been able to recognize certain behavioural and physical changes related to the reproductive state of cattle 1,2.

Among ruminants, such as bovines, as well as in other species, during the oestrus stage a somewhat translucent and relatively sticky aqueous substance can be seen to copiously come out from the female reproductive tract, one of the signs that has been recognized, from ancient times, as the beginning of sexual receptivity heat 1,2.

Such secretion, known as cervical mucus, exerts several physiological functions that are critical for the development of the reproductive process in the cow, the same as in other animals in which this fluid is produced.

When a drop of cervical mucus collected at oestrus is deposited on a transparent surface and allowed to dry at room temperature, the mucus tends to crystallize in highly arranged geometric patterns, characterized mainly by arborescent morphologies, among other arrangements 1, Assessment of the properties of cervical mucus throughout the oestrous cycle shows that the extent of the crystallization phenomenon reaches a maximum at oestrus in comparison to any other stage of the cycle 4.

This is mainly due to raised levels of oestrogens in this stage which are exerting their effects on the cervical mucosa, and, therefore, changing the mucus features. This explains why the assessment of mucus crystallization has been suggested by some researchers as a useful tool to determine the onset of sexual receptivity in cows 1,8,9 and other animals 10, The objective of this review article is to discuss the main aspects of the phenomenon of mucus crystallization, with special interest in the characteristics of the crystalline patterns observed in bovine cervical secretion.

Cervical mucus is produced by mucus-secreting cells that line the grooves and folds resembling 'blind-ended crypts,' present in the cervical epithelium Secreted more copiously at oestrus, mucus volume can reach up to mL. Among the soluble substances are proteins, such as lactoferrin 16 , immunoglobulins, several enzymes, e. On the other hand, the non-soluble fraction consists of high-molecular-mass glycoproteins known as mucins 15, , mainly those classified as secreted gel-forming mucins These are highly glycosylated proteins and probably constitute the main factor responsible for the rheological properties of mucus, such as its variable elasticity, viscosity and spinnbarkeit, among others The cyclical variations in the levels of oestrogens and progesterone have a marked effect on bovine cervical mucus.

In general, these hormones exert their influence by directly acting on secreting endocervical cells, mainly via mechanisms mediated by classical steroid receptors 27 ; as a consequence, the composition, the physicochemical and structural properties, and the rheological attributes of the cervical secretion are changed Regarding this, it has been shown that mucus water content varies along the cycle, increasing at oestrus 29,30 , due mainly to the rise in oestradiol levels observed in this stage.

Also, mucin types are differentially expressed during the stages of the oestrous cycle 12,24 , a change also related to fluctuations in sex steroid hormones. Bovine cervical mucus has several important functions in the reproductive process, among which are:.

The cervical secretion protects the bovine reproductive tract by maintaining the epithelial surfaces moist and lubricated. This is due to the high level of hydration that characterizes this gel 15 , since mucins are capable of binding large volumes of water 22, Cervical mucus takes part in sperm selection and transport, being the first medium spermatozoa must go through when ascending to the site of fertilization 15,32, During the periovulatory period, mucus secretion increases, becoming less viscous and more hydrated, facilitating the ascent of spermatozoa 15,32, Also, in this period, the mucus structure would facilitate the movement of normal spermatozoa and inhibit the ascent of gametes with morphological alterations, acting as a selective filter On the other hand, in the luteal phase, the amount and hydration of the secreted mucus decreases while its viscosity increases, preventing spermatozoa migration.

According to Becher et al. The cervical secretion constitutes an immune barrier that inhibits the ascent and colonization of microorganisms, since some of the compounds present in mucus can inhibit the penetration and proliferation of microbes During pregnancy, cervical mucus protects the uterus from environmental noxious agents, since during this period mucus forms a highly viscous barrier known as cervical mucus plug 31, A number of substances have been identified in the fluids of a cow's reproductive tract, among which there are sex steroid hormones These are also present in cervical mucus and probably modulate the acrosome reaction acrosomal exocytosis , as it has been proposed for human cervical mucus In cows, this idea is also supported by evidence obtained by using scanning electron microscopy when studying cervical sections in follicular phase, in which spermatozoa with intact acrosomal membranes in some mucus-filled luminal regions were observed However, further studies are needed to elucidate the role exerted by sex steroid hormones present in bovine cervical mucus on acrosomal exocytosis.

In general terms, crystallization can be defined as the process through which a component of a liquid solution changes to its solid phase, tending to separate from the solution and to precipitate in the form of crystals.

Crystallization constitutes a means to reach a more stable, lower energy state from a metastable solution by reducing the solute concentration Crystallization is produced by molecular aggregation leading to the formation of crystalline nuclei nucleation , with the subsequent growth of those nuclei. Therefore, nucleation is the precursor of crystalline growth. The process of crystallization is not exclusive to cervical secretion; it is present in a number of other biological secretions, for example, human and bovine saliva Secretions able to crystallize are characterized by containing mucoproteins e.

In fact, NaCl is the main salt found in cervical mucus, providing the mucus with ionic strength In general, cervical mucus crystallization has been studied by spreading mucus sample drops onto a glass slide so that, after drying at room temperature, a smear or film forms on the slide. This can be observed without staining by using a standard light microscope 1,4,5, The first to report on cervical secretion crystallization was Papanicolaou , who focused on the fern-like arrangements observable on women's mucus, suggesting that this phenomenon could be used as a predictor of ovulation As a result of the crystalline shapes that were observed, from then on, cervical mucus crystallization has also been called an 'arborization' or 'ferning phenomenon' Figure 1.

Garm and Skjerven 46 studied crystallization in the cervical mucus of cattle, finding abundant fernlike crystals during the follicular phase, which disappeared during the luteal phase and were non-detectable in the early stages of pregnancy. Later, several researchers determined that the highest arborization occurred at the onset of, or during, oestrus 1,47, In this regard, Abusineina 1 stated that the study of cervical mucus in relation to the presence or absence of crystallization, and the type of crystallization observed, is an indicator of the stage of oestrous cycle and the day of ovulation; therefore, it is useful to confirm clinical findings as well as for experimental applications.

As the proportion of salts in the dry residue starts to decrease, so does the water content. This change leads to a decrease in the arborizations observed in the dried mucus sample. In relation to this, Noonan et al. The concentration of dry matter in cervical mucus reached a minimum at oestrus and a peak at mid-cycle, while mucus ferning appeared at oestrus to a greater extent than at any other stage of the oestrous cycle. Also, as for other properties of cervical mucus, variations in the occurrence of crystallization during the oestrous cycle are equally due to the changing levels of sex steroid hormones.

In general, oestrogens are considered to promote crystallization, while progesterone decreases it In this regard, it has been proposed that the higher occurrence of arborizations at oestrus depends on oestrogen dominance during the follicular phase Oestrogens would cause an increase in the ferning phenomenon through mechanisms that stimulate the electrolyte metabolism in the cervical epithelium 50, On the other hand, in the luteal phase, increased levels of progesterone would counteract the effects of oestrogens on the cervix, explaining the decrease in arborizations 50,52 , a fact that is in agreement with the inhibitor effect on crystals previously reported for this hormone The influence exerted by these sex steroids coincides with the observation made by Elstein 54 , who states that, among the many attributes of cervical mucus, arborization is, without a doubt, one of the most sensitive to variations in the levels of sex steroids.

Nowadays, it is well-known that crystallization constitutes a useful property for studying both cervical mucus and the reproductive cycle 5, On the other hand, it is also worth mentioning that the crystallization of cattle saliva undergoes changes during the oestrous cycle, and hence the analysis of such crystallization can help in the diagnosis of early pregnancy To our knowledge, the first model to classify crystallization patterns of bovine cervical mucus was proposed by Abusineina 1 , who divided the observed arrangements into three types.

Type A corresponds to the crystallization observed when the mucus is translucent, acellular, elastic and easily obtained from the cervix. Under a light microscope, Type A crystallization is characterized by the presence of a long, thin stem main axis , which may be straight, waving or curved. From such a stem, well-defined venations of variable length protrude with tiny subvenations Figure 2A.

This type of crystallization would be associated with ovulation and generated as a consequence of high oestrogen levels 1. Type B crystallization corresponds to that observed when mucus is semi-translucent, elastic and easily obtained from the cervix. When observed under a light microscope, this type is nearest in shape to a fern frond 1. Venations and subvenations are well-defined and easy to observe Figure 2B. Type C corresponds to the crystallization of opaque mucus, evidencing cellularity and being difficult to obtain from the cervix.

When observed using light microscopy, Type C crystallization is irregular and its fern-like morphology is atypical. The central axis is short, with or without venations and subvenations, which are irregular 1 Figure 2C.

Some disperse linear crystalline patterns can be found, either cruciform Figure 2D or stellate. Another model for the classification of bovine cervical mucus at oestrus was proposed by Bishnoi et al.

Score 0 corresponds to the absence of crystalline formations Figure 3A. Score 1 is assigned to the formation of atypical crystals only Figure 3B. Score 2 is assigned when many atypical and a few typical fern-like crystals are observed Figure 3C. Score 3 represents the formation of many typical fern-like crystals and a few atypical crystals Figure 3D.

Finally, score 4 is given to patterns showing the typical fern frond crystal arrangements Figure 3E. A well-known classification model for the crystallization of cervical mucus of the periovulatory period was reported by Odeblad 57 and later validated by other investigations 58, Initially proposed for women's cervical secretion 57 , it is interesting and worth mentioning that, when studying bovine cervical mucus according to the classification proposed by Odeblad, geometrical crystallizations very similar to those obtained for human cervical mucus are observed 5,7.

The types and subtypes proposed by Odeblad, and observable in bovines, are: Type S Crystallization: its morphology resembles straight lines that tend towards a parallel arrangement Figure 4. This crystalline pattern is evidently fern-like Figure 6A.

Subtype P6 B has a very attractive geometry, resembling a star, with a central nucleus from which six well-defined axes protrude 7. In general, this subtype has been found to form somewhat larger crystalline units than other subtypes of P mucus 58 ; also, subtype P6 B in humans would be linked to the fertility peak Subtype Pa has a crystallization centre from which multiple branchings irradiate in all directions Figure 6C.

Lastly, another subtype corresponds to mucus Pt, which does not have such an orderly arrangement like the aforementioned subtypes; the crystals appear to be more disperse and not always joined Figure 6D , as observed in women 58, Similarly, it is worth mentioning that, when observed under light microscopy, bovine cervical mucus also shows some crystalline arrangements that cannot be satisfactorily categorized into any of the previously described models Figure 7.

Finally, certain arboriform crystallizations of heifer cervical mucus have been reported to show a fractal-like organization, i. These fractal patterns have also been proposed for human mucus 62 , even though their biological significance is yet to be explained.

During the oestrous cycle, and especially at oestrus, it is possible to identify several geometric arrangements for the crystalline patterns of bovine cervical mucus; this fact has made it possible to propose the previously described classifications. The reason for the existence of such types of crystallization has not been completely elucidated, but considering that bovine cervical epithelium could be comprised of different secretory regions 12,63 , those crystalline patterns would be result of the differential influence exerted by raised levels of oestrogens at oestrus on such regions.

Thus, cervical mucus is probably a heterogeneous entity formed by the admixture of several subtypes of secretion 59,64,65 , with proportions varying in the periovulatory period and, to a lesser extent, during other stages of the cycle. This fact would explain the existence of different morphological types and subtypes of mucus crystallization; other possible underlying causes are variations in salt content as a result of modifications in electrolyte metabolism at the level of the cervix and water content, as well as the arrangement and type of mucin present in cervical mucus due to changes in levels of sex steroid hormones.

The study of the crystallizations present in bovine cervical mucus at oestrus and other stages could lead to a deeper understanding of bovine reproductive physiology, both in physiological and pathophysiological conditions.