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. Trophoblast 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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