INTRODUCTION High reproductive efficiency is a prerequisite for life time production in dairy animals

INTRODUCTION
High reproductive efficiency is a prerequisite for life time production in dairy animals; hence an early and accurate diagnosis of reproductive dysfunction is crucial for better reproductive management in livestock. Pregnancy diagnosis is an important part of reproductive management in efficient farm operation. Hence an early and accurate diagnosis is essential because
? It helps in identification of non-pregnant animals which can be treated or rebred earlier
? It is an important factor to shorten the calving interval
? To increase milk production
? To certify the animals for purpose of sale
? To reduce wastage in breeding programme
? To increase overall profit
METHODS OF PREGNANCY DIAGNOSIS
Several methods of pregnancy diagnosis are being practiced in bovines but none till date qualified as an ideal diagnosis method. The ideal pregnancy diagnosis method should have sensitivity, specificity, simple and inexpensive to conduct under field conditions.
Pregnancy diagnosis is broadly classified into 2 types
1. Direct method
2. Indirect methods
DIRECT METHODS
a) Non return to estrus
When an animal is mated and when it does not return to estrus, the owner thinks animals has been pregnant. This happens during pregnancy, where the conceptus inhibits regression of corpus leuteum and prevents animal from returning to estrus but however many times the animal does not return to estrus because of reason other than pregnancy.
b) Per rectal palpation
It is oldest, easiest, cheapest method of pregnancy diagnosis carried out even today. This method is techniques of choice taught to veterinary graduates. This method is traditionally carried out at 40 to 60 days of gestation but early detection ie on 30 days depends on skill of examiner and his prior experience.
c) Ultrasonography
Ultrasonography can be adopted from day28-30 after AI. It is accurate and efficient technique for early pregnancy diagnosis. The main advantage is it can give accurate diagnosis and minimize the incidence of palpation induced abortions and it also provides additional information on ovarian structure, age, sex, viability of fetus and twins identification. But machines are expensive and require an efficient operator. The most commonly used machines today are B mode real time range from 3.5-7.5 mHz.
INDIRECT METHOD
a. Progesterone hormone assay
Measurement of progesterone is an indirect method of pregnancy detection in livestock species. Presence of conceptus in uterus extends the life of CL by preventing luteolytic mechanism and thus maintaining the normal functional characteristic of CL required for implantation and embryonic development.
Studies indicated that progesterone in milk remains elevated up to day 21 after fertilization and beyond. These high levels between day 18 and 24 after insemination form basis of establishment of pregnancy in cattle, but however if the cow is not pregnant it will be in diestrum phase with active CL producing progesterone that can result in false positive results.

b. Estrone sulphate
It is conjugated estrogen that can used to diagnose pregnancy. It is present in the bovine placentomes and fetal fluids and maternal plasma, milk. In cows concentration of estrone sulphate increase from about day 60 of gestation but reliable pregnancy detection is possible only after 100 day of gestation hence this test can only detect late pregnancy.

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c. Milk ejection test
This test is directly correlated to plasma progesterone profiles. The intrajugular administration of subluteolytic dose (250-500 micrograms) of PGF2? day18-22 after mating releases luteal oxytocin. The animal in pregnant shows increase in pressure in milk ejection and milk volume in comparison to non-pregnant animals.
CANDIADTE MOLECULE ASSOCIATED WITH EARLY PREGNANCY IN CATTLE
A. Early conception factor
It is also known as early pregnancy factor (an immunosuppressive glycoprotein present in sera of pregnant animals that can be detected within 24 to 48 hours of fertilization and disappeared within 24 to 48 hours after death or removal of embryo. A chemical test for ECF was proposed and marketed in 1990. But different studies found that kit was unreliable for pregnancy detection.

B. Pregnancy associated glycoprotein
The mono and bi nucleate cells of embryonic trophectoderm migrate and fuse with the epithelial lining of uterus at the moment where the conceptus becomes attached to uterine wall and formation of placentome begins. These cells are unique because they secrete placental proteins into capillary beds of uterus that connects to peripheral circulation of cow. One type of protein secreted in PAG (also known as pregnancy specific protein B). These molecules appear in maternal circulation and can be determined with accuracy from day 29 to 30 post breeding. Simple ELISA technique has been developed to detect PAG molecule in serum of cows.
C. Interferon tau
During the third week the embryo undergoes transition from spherical to filamentous form. During this transition the embryo produces a protein between 16 to 25 days to signal the mother and to establish pregnancy. Primary function of interferon tau is to prevent uterine luteolytic mechanism to ensure maintenance of CL for pregnancy. The presence of interferon tau is estimated by measuring IFN-tau stimulated genes (ISG) expression in circulating leucocytes, examples of ISG are
i. 2-5 oligodenylate synthetase 1
ii. Myxovirus resistance gene 2
iii. Interferon stimulated gene protein
These respective genes were assayed by using reverse transcriptase polymerase chain reaction (RTPCR)
D. Circulating nucleic acids
These are cell free or membrane wrapped DNA and/ or RNA circulating in blood. These can be usefull for the detection of early pregnancy by using quantitative real time PCR .one of the most promising candidates is circulating microRNAs (miRNA).