Cervical Alterations During Pregnancy in Small Ruminants

October 14, 2009 by Admin  
Filed under Science

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Dr. Pankaj Goswami asked:


Cervical alterations during pregnancy in Small Ruminants

 

P. Goswami and G. M. Wani

Directorate of Extension Education,

SKUAST-K, Shalimar Srinagar

 

 

The cervix uteri is a thick walled fibromascular tube connecting the body of the uterus and ******. It a muscular organ composed of connective tissues predominantly elastic fibres. In the non pregnant ewes, the cervical canal is impassable except during oestrous. Five or six hard prominences within the canal assist the sphincter effect of the cervix (Nickel, Schummer & Seiferle, 1973). During the course of gestration, the length of cervix increases and in late pregnancy the wall becomes thicker, with an overall increase in the compliances of the tissues (Cloete, 1939; Abusineina, 1969). This may be related to disaggregation of densely packed collagen fibre in the cervix of pre-partum ewe. This paper will focus some of the important physical and microscopical changes occurred in the cervix of small ruminants during pregnancy

 

Anatomy of the cervix:

The cervix separates the uterus from the ******. During pregnancy, it seals and protects the embryo and fetus from the external environment. The gross and microscopic anatomy of the cervix has been studied by various workers. The casts of the inside of the cervical lumen shows its convulated structure consisting five to six circular folds and the second fold being eccentric  to the other concentric folds and acting as physiological barrier. The cervical fold in small ruminants varies from five to six folds. In cow four large circular and 15-25 longitudinal primary folds each with many secondary and tertiary folds are present. Cervical mucosa is generally characterized by longitudinal primary fold and most of which maintained continuity throughout the cervix.  Superimposed on these secondary folds which is varied in length and depth. Abundant shallow uniformity and parallel longitudinal grooves covers all surface.

 

 

Morphological changes in the cervix:

Three major changes generally observe in cervix during pregnancy. These are described as growth (physical increase in length and breadth). Softening (changes in tensile properties) and dilation to allow passage of the foetus. The study carried out by different workers showed that ovine cervix shows an increase in width and length in the later stages of pregnancy. The analysis of the constituency of cervices shows increase softening from the mid pregnancy and there after firmness of cervix losses.

A small increase in the degree of hydration of the cervix or dry weight at different gestrational stages has been reported by Fosang et. al. (1994), ward 1968. This may be due to increased tissue mass rather than increase in size of water content. However some author reported no significant changes/differences in water content of the cervices from non pregnant to pregnant animals. The physical chemical and histological properties of cervix are constant throughout the length of cervix. However, Basset (1958) reported morphological changes in the fibroblast of the broad and sacro-iliac ligament by the 60th day of pregnancy but this information is not supported on ultrastructural studies.

 

Light Microscopical changes

Morphologically the most prominent feature of non pregnant cervix is heavy, densely packed collagen fibre interspersed with fibroblast (fig.3 ). Small blood vessels are present throughout the depth of the tissue, but most numerous in deepest layer. Smooth muscle bundle are running both longitudinally and transversely in the middle and deeper layer. The figure represents a wall of non pregnant cervix. The lining epithelium is low columnar and secrets neutral  mucin. The sub epithelial connective tissue is vascular and contains variety of cells including eosinophil, macrophages, mast cells and plasma cells. The greater proportion of cervical wall is composed of dense fibrous connective tissue consisting of compactly arranged collagen fibre with some fibrocytes and occasional fibroblast embeds in sparse ground substance. The individually arranged smooth muscle fibre forms an incomplete muscularies of which the outer fibre is longer and more prominent than inner fibre. The electron microscopically the collagen fibre shows very compact in arrangement and the scarcity of the ground substance and the presence of fibrocytes. Fosang et. al . opined that there is no significant changes observe between proximal, middle and distal portion of cervix irrespective of stain used. The best stain normally use for differentiation between collagen fibre and the smooth muscle bundle are Massons’ Trichrome stain, where the alignment of the collagen fibre along with villi shows projecting towards lumen. In general collagen fibre are large and closely spaced and are organized either longitudinally or obliquely. Section stained with Toludine Blue stain revels metachromatic staining along the collagen fibrils with strong staining of epithelial cells associated mucus. The morphological changes donot become apparent until quite late in the gestration period. The description of non pregnant cervix applied equally to the connective tissues observed in the early stages of pregnancy even to 100 days.  

                                                             

Fig. Pregnant cervix showing

      

Fig: Dense Collagen fibre, inner circular & longitudinal muscular layer with epithelium H&E 4X

Fig. Central cervix Transverse section Loosening of epithelium and collagens layer H&E  (pregnant)

 

 

 

 

 

The histological section at 100 days of pregnancy revels no virtually distinguish alteration from that of non pregnant cervix (Calder et. al).  The tall columnar cervical epitheliums are the only changes represents in pregnancy and the secretions are a mixture of acid and neutral mucin. Acidity increases with the pregnancy age. Tissue breakdown and destruction of collagen networks is evident at 140 days of pregnancy. The cells are more widely spaced (empty area) and the collagen fibre losing their organization exposing smooth muscle cells. This can be best seen with Massons’ Trichrome. The infiltrating cell at this stage are lymphocytes and monocytes and few eosinophils. In late gestration increased fibroblast activity, smooth muscle hypertrophy, vascular edema and dissolution of collagen fibre bundle are reported by various worker. These findings contrasted with the rigid fibromuscular tissue observe in the non pregnant animals. The appearance of thinner fibre and empty areas between fibres in late pregnancy is lead to decrease concentrations of hydroxyproline in tissue. Collagen fibre dissolution in pregnant cervix has been extensively reported in several species and many authors have reported that active collagenolysis occurring during pregnancy may be the underlying mechanism of cervical softening. Ellowed et al (1981) have shown that ovine cervical explants produce both latent and active collagenase activity, with greater yields of activity in parturient tissue compared with the late pregnancy after 3-5 days in culture. Inflammatory cells invading cervix towards late gestration provide a potential source of collagenase and neutral protinease activity. Eosinophils also have been described as potential bearer of specific collagenase which may be responsible for collagen catabolism (Basset, 1972). At the term the disruption of collagen fibre are more even pro
nounced with virtually no large fibre remaining. In Haematoxyline & Eosin stain sectioned it sometimes appears very little or no collagen at all. But very little and small fibrils arranging random pattern are seen in Massons’ Trichrome stains. In this stage there is heavy infiltration of inflammatory cells among which eosinophils predominant. An area of haemorrhage is also a constant finding along with infiltrating cells. In late pregnancy there is complete network of subepithelial capillaries with a marked increase in the size of the vessels in the outer part of the cervical wall.

 

Ultrastructure feature:

Ultrastructuraly, non pregnant cervix reveals the typical dense connective tissue with collagen aggravated in closely packed fascicles and fibrocytes embedded in sparse ground substance. The ultrastructural characteristic in late pregnancy are presence of rough endoplasmic reticulum, mitochondria,plasmalemmal vesicle and extensive branching of individual fibres in contrast to the absence of these feature in muscle fibres of the non-pregnant cervix. This description is also similar to early pregnancy stage. The ultrastructural analyses of the cervical connective tissue reflects active changes in tissues, with a reorganization of the cervix prior to the functional changes at parturition.

 

 

 

Changes in collagen concentration:

The biochemical analysis of hydroxyproline in tissue can be used for collagen concentrations. Study carried out by Regassa et al. (1983) shows the total collagen content of cervix at all stages of pregnancy is significantly greater than that of caruncular mean and the intercaruncular areas. The concentration of hydroxyproline is not changed in cervix during Ist trimester of pregnancy. However the concentration of hydroxyproline progressively decreases at days 100, 140 days and in post partum tissues as compared to the non pregnant tissue(Fosang et. al 1984). The concentration is same between proximal, middle or distal region of the pregnant and non pregnant cervix.

In conclusion it is summarized that uterine cervix of small ruminants became softer during the pregnancy and that some associated changes first appear in early gestartion. There is no significant changes in water content through pregnancy although light increases is associated with cervical size and softening of the tissue. Physical and histological properties are identical in all section along the length of cervix. The changes associated with increasing length of gestration are absolute increase in width and length, relative increases in fibroblasts, smooth muscle and softening; relative decreases in collagen and fibrocytes. But increased vascularisation without any white cell infiltration of the tissue is specifically associated with late gestration.

 

 

REFERENCES

Abusineina M.E. (1969) Effect of pregnancy on the dimendions and weight of the cervix uteri of sheep. British Vet. J 125, 21-24

 

Amanda J. Fosang, Christopher J. H. Vivien S., Dennis A. L. and Geoffery D. T. (1984) pregnancy related changes in connective tissue of ovine cervix. Biology of reproduction 30, 1223-1225

 

Aughey, E, Munro, C. D., Calder, A. A., Coutts, J R. T. & Fleming, R (1981). The histology and ultrastructure of the pregnant sheep cervix uteri. J. of Anatomy 132, 448

 

Basset, E. G. (1958) Gestational changes in connective tissue. Nature 181, 196-197

 

Cloete, J.H.L. (1939) prenatal growth in the merino sheep onderstepoort journal  of veterinary science & animal industry 13, 417-543

 

Calder A.A., Aughey E. Coutts J. Fleming R and Munors C.(1983) Changes pattern of cervix on pregnancy J. Anat (1983) 136, 2 389-399

 

Ellwood D.A., Anderson, ABM, Mitchell and Turnbill A.C. (1981) Prostanoids, collagenase and cervical softening in sheep. Am. J. Obst. Gyneol. 10:281-287

 

Hollingsworth, M. (1981) Softening of rat cervix during pregnancy. In the cervix in pregnancy and labour- clinical and biochemical investigations (ed. D. A. Ellwood & A.B.M. Anderson) pp.13-33 Edinburg

 

K. June Mullins, R. G. Saacke (1988) Study of the functional anatomy of bovine cervical mucosa with special reference to mucus secretion and ***** transport Journal of Reproduction and Fertility (1979) 57 261-266

  

Karen Sohan , Rebecca Wiggins and Peter Soothill (1999), Cervical Physiology in pregnancy and labour. Foetal and Maternal Medicine review 11: 135-141 Cambridge

 

 More J (1984) Anatomy and Histology of the cervix uteri of ewe: A new insight Acta. Anat (basal) 120 (3). 156-9

 

Nickel, R., Schummer A. & Seiferle E., (1981) The viscera of domestic animals pp.358 and 361 berlin verlag Paul Pavey.

 

Regassa F. and Noakes D. E.(1983) Changes in the weight, collagen concentration and content of the uterus and cervix of ewe during pregnancy. J Biology 73, 221-25

 

 



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Early Pregnancy Diagnosis in Ruminants

May 6, 2009 by Admin  
Filed under Education

Prof G M Wani asked:


PREGNANCY DIAGNOSIS IN ruminants

1 Introduction

Rectal palptation in small ruminants is of little value due to the size of the pelvis. (Wani, 1981). The caudal artery monitoring, bloatment, non-return to oestrus, udder development and other tests tried have had little success, (Wani & Sahni,1980). The more recent interest in early pregnancy 3. diagnosis of small ruminants is of academic and economic importance (Mellado,2003). A highly valued zygote or embryo when transferred to a less valued surrogate mother (recipient) needs to be closely monitored and the early detection of conception helps in repeated use of baren females. Proper management of pregnant animals also prevents embryonic losses. The method applied should be safe to both offspring and dam and needs to be cheap and easily applied. A review of various methods and techniques used for early pregnancy diagnosis in small ruminants. (sheep and goats) is presented.

.2 Early Signs of Pregnancy

2.1. Maintenance of a functional corpus luteum

It was evident that conception prolongs the life of the CL and prolongation and maintenance of a functional CL is triggered by the developing conceptus. These signals ensure the maintenance of the structural integrity of the CL. Corpus luteum produces progesterone, which maintains the uterine endometrium in a state permitting embryonic development, implantation and foetal-placental development (wani,1984b) . The formation and regression of the corpus luteum (CL) in Muzzaffarinagri ewes and Jamunapari goats was monitored at 3 days intervals for an entire oestruous cycle. Laparotomy and laparoscopic methods were used in these experiments.

The Endometrium undergoes tissue remodeling. This change in Extra cellular Matrix (ECM ) components is needed for successful implantation. Cytokinens 8,18 and 19 have been detected in the caprine endometrium during early pregnancy using immunofluorescence. Thus the presence of these cytokinen at approximately day 15 post conception is indicative of pregnancy in goats.

The implantation process in goats starts around day 18 post mating. During this phase intense type I collagen staining was detected throughout the uterine caruncular and intracaruncular stroma. For embryonic trophoblastic adhesions with endometrium, local control of protease activity is suggested. (Guillomot, 1999).

The earliest signs of pregnancy is the non-regression of the cyclic CL, which can be observed by the following methods:

i. Laparoscopy and Laparotomy approximately day 18-25 post mating. (Wani, 1982, 1988, 1984b,Wani & Buchoo, 1990, Wani & Buchoo, 1993, Cuellar et al, 1990, Wani et al, 2003).

ii. Serum Progesterone values higher than 1 ng/ml e.g 2 to 3 ng/ml. (Wani, 1989; Shreif, 1997, Boscas et al, 2003, Al-Merestani et al, 1999, Zarkawiet et al, 1999). Diagnosis of Pregnancy accurately (100%) predicted on the basis of serum progesterone P4 values around 17-19 days post mating .

iii. Pregnancy associated ovine glycoproteins recorded approximately post mating indicate pregnancy in sheep. (Karen et al, 2003; Verberckmoes, et al, 2004) or secretion of 17 & 22-24 K Da proteins on day 17 post mating in the caprine conceptus. (Guillomot et al, 1998).

iv. Non-return to oestrus (Mellado, 2003)

Some of the other early pregnancy signs detected by various methods are set out in Table 1.

3 Non-rejection of early conceptus

Progesterone maintains the uterine endometrium in a state which allows for embryonic development, implantation and foetal placental development. Details of foetomaternal relationships have been described (Mufti, 1997, Mufti et al, 2000)and are shown illustrated in Fig 1to 5. The presence of an early conceptus prolongs the life of corpus- luteum. These pregnancy signals are secreted as proteins. (Heap et al, 1990). Some of these proteins have been identified as ovine Trophablast protein I (OTP-1) in sheep which prevents the release of PGF2 alpha and thus helps in the maintenance of the corpus luteum. In cyclic ewes (non-pregnant) PGF2 alpha pulses are released in response to oxytocin with receptors being in the endometrium. The earliest signal of pregnancy is detected by a marked reduction in the endometrial oxytocin receptor numbers. The OTP-1 may inhibit synthesis of endometrial receptors for oestrogen and oxytocin. This possibly prevents luteolysis and maintains the dominance of theuterus by progesterone which is pre-requisite for the establishment and maintenance of pregnancy. (Bretzlaft and Romano, 2001; Wani, 1996; Ala cam et al, 1988).

The expression of progesterone receptors (PR) in the caprine uterus markedly increases during the peri-implantation period and estrogen –(ER) receptors do not increase in relation to PR, thus signaling the non-rejection of the early conceptus. (Flores et al, 2001). Progesterone in milk too can be found during early fertilization and conception (Cough et al, 1989).

Caprine H-type I antigen expression is unregulated during peri-implantation and progesterone P4 level stimulate it. It may be a useful marker to signal uterine preparations for receiving and retaining pregnancy in goats. (Powell et al, 2000). The caprine pregnancy related glycoprotein (Ca PAG) may help the conceptus to develop and is found around 18-19 day post mating . (Garbayo et al 2000). Endometrial tissue the undergoes remodeling to retain the conceptus in gravid small ruminant females. (Guillomot, 1999)

The dephosphorylated state of caprine uterine myocin in early pregnancy may help the conceptus to grow. Changes in the expression of native myocin, myosin heavy chains (MHCS) and myosin light chains (MLCS) were observed. (Kumar and Katoch, 1997).

For the development of the blastocyst, a proper uterine environment is essential. Besides the maintenance of the corpus luteum, production and availability of progesterone, the non-rejection of conceptus (blastocyst) is another critical feature of this period. The embryo produces interferons (embryo-IFN). This embryo IFN is homologous with – interfersons ( ? –IFN) and Ovine Trophoblast Interferons (OTI) of early pregnancy.

Purified OTP and recombinant OTP (r-oTP) produced in yeast exhibit antiviral activity and these r-OTP and OTP inhibit the release of endometrial PGF2 ? . This helps in the non-regression of the CL and indirectly maintains the early conceptus. Intra uterine r-OTP administered at a dose of 340 µg/ day for a week maintained the C.L in cyclic ewes for a month or so of . The inter- oestruos interval in 80% of the ewes was about a month or more. This dose r-OTP was as a effective as 14-16 day old conceptus. OTP was found to be immunosuppressive in several in-vitro and in-vivo assays. An assay on phytohaemagglutinin A revealed both OTP and r-OTP to be immunosuppressive. This was further verified by the inhibitory activity of r-OTP in Graft Versus Host Reaction. (GVH assays). Trophoblast interferons play a strategic role in the prevention of early pregnancy loss as it inhibits CD + blastogenesis. The role of CD + cells and as helper T lymphocytes and delayed+ Type hyper sensitivity mediators (DTHS) would explain this immuno- suppressive rate of OTP. (ILeri et al, 1996; Karen et al, 2003; Wani, 1996).

3.4 Oestrogen: – Pregesterone ratio (E:P ratio)



The role of oxytocin in inducing uterine PGF2 alpha was discussed earlier. However, the release of PGF2 under the action of oxytocin depends on or is controlled by progesterone and oestradiol. It was further indicated that ewes with a high E:P ratio may generate stronger luteolytic signals. It was demonstrated that low progesterone and high oestradiol combination record the largest and sustained increase in PGF2 alpha following oxytocin injection. Trophobla
st interferons act locally to suppress the uterine oxytocin receptors in sheep.(Karen et al, 2003 ).

5 Maternal recognition of pregnancy

The maternal recognition of pregnancy in sheep and cattle is centered around the production by the trophoblast of type I x interferon (tINF). This tIFN then suppresses uterine oxytocin receptor concentrations (OTr). The oxytocin receptor (OTr) occupancy is associated with oxytocin induced PGF2 alpha release. OTr inhibition may represent the principal antiluteolytic mechanism of tIFN and secretion of the conceptus secretory proteins or bovine recombinant IFN to the uterus reduces OTr. Concentrations in intact and ovarectionized steroid treated ewes . A relationship between the conceptus secretory proteins and the metabolic products and those in the peripheral blood of the dam exists. ( Mufti; 1996; Mufti et al, 2000). There are conflicting reports making the action of oestradiol on oxytocin receptor concentration. (Powell et al, 2000). Trophoblastic cells contain interferon on day 14-17 after mating. During maternal recognition of pregnancy goat interferon was detected on day 18 post mating, its absence signifies pregnancy maintenance has been taken over by the corpus luteum. Thus a very thin line exists between maternal recognition of pregnancy and its maintenance or sustenance by the CL. (Gillomot et al, 1998).

6 The Reliability Pregnancy tests

Various methods used for correctly predicting pregnancy in sheep and goats during gestation have been summarized in Table 2. The accuracy varies from 70 to100% with different ultrasonic equipment. Different models as well as principles involved have been extensively reviewed (Wani, 1991; Wani et al,1998) and other methods of pregnancy detection during this stage e.g serum progesterone determination, vaginal cytology, laparotomy, estrone sulphate are summarized (Table-2). Various techniques were also evaluated in assessing mid-gestation. The various pregnancy signs as quoted by

various researchers using ultrasonography are summarized in Table 3. Of late certain anatomical features in the live, developing conceptus in vivo have been reported. This is reviewed and a summary is presented (Table 4). Various live foetal measurements like Biparietal diameter, Amniotic vesicle diameter, foetal radius and Tibia lengths are reviewed and shown (Table 5). Various pregnancy related images, histological sections and morphology of endometeruim have recently be published (Wani et al 2007, 2006 abc) where images are presented 6-15

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