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Pregnancy markers for early pregnancy diagnosis

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“Detection of the pregnant/ non-pregnant cow/ buffalo, heifer, as soon as possible after a successful/ unsuccessful insemination, is important to ensure good reproductive and / productive performance in dairy and beef animals”

-Omics revolution and integration of all -omics sciences with a systems approach could help find solutions towards finding early pregnancy biomarkers.

Pregnancy markers for early pregnancy diagnosis

  1. 1. “Detection of the pregnant/ non-pregnant cow/ buffalo, heifer, as soon as possible after a successful/ unsuccessful insemination, is important to ensure good reproductive and / productive performance in dairy and beef animals”
  2. 2. ∗ Ovulation : 20-36 h of 1st detection of estrus ∗ Fertilization : within hours of ovulation ∗ Transport to Uterine horn : 3½ to 4 days ∗ Hatching: Day 7 ∗ Maternal recognition of pregnancy: Day 14-19 ∗ Implantation : Starts on Day 22 - completed by Day 40 ∗ Pregnancy losses ∗ Fertilization losses : 5-10% ∗ Early embryonic mortality : 10-20% ∗ Late embryonic losses : very few (<5%) ∗ Post implantation losses : rare ∗ Most reliable indication of successful pregnancy: implantation i.e. Day 40+ Major Landmarks: Bovine/ Buffalo Early Pregnancy
  3. 3. time……the commencement of prenatal care Changes in management, feeding and health care……to promote a healthy pregnancy/ early pregnancy termination if so desired improve reproductive efficiency and pregnancy rate by decreasing the interval between AI services increasing AI service rate Economic considerations…. to farmers, dairy entrepreneurs, policy makers Early pregnancy detection: why?
  4. 4. ∗ Cattle and buffalo husbandry…….. Taking shape of industry ∗ Intensification of reproductive management in dairy/ beef herds and the implementation of AI are creating the need for more accurate and timely diagnoses of pregnancy ∗ At the same time, there is a shortage of large animal veterinarians/ para-veterinarians in many regions…..reasons!!! ∗ Shortage………growing realization of pressure on limited human resource to complete a large number of pregnancy diagnoses ∗ In some cases, there is the desire to perform the pregnancy exams sooner after insemination so that non-pregnant cattle can be identified earlier and resynchronized for a second AI Early pregnancy detection: why?
  5. 5. ∗ The human pregnancy produces copious amount of a hormone called hCG that passes into the urine and can be detected by a simple lateral flow ELISA test (Fletcher, 1986) ∗ Unfortunately cows/buffaloes do not make bovine chorionic gonadotropin (or any such molecule that is readily detectable in the urine…..till now) ∗ Outcome………so a simple test that is similar to the human test is not available!! If cattle & buffalo were humans then the solution would be simple ☻
  6. 6. “It creates an opportunity for the identification of pregnancy markers for application in chemical pregnancy testing (for example, blood/ urine/ saliva/ other non- invasive biological tests for pregnancy)”
  7. 7. • Direct methods: Rectal/ Trans-rectal / Per-rectal palpation, Ultrasonography • skill and experience / instrumentation • Indirect methods • Immunoassays based on Progesterone and PAGs • Sensitivity, Specificity and Accuracy • Pregnancy marker molecules??? • prerequisites • specifically up- or down-regulated • least affected due to non-animal factors • present in easily accessible body fluids • expressed over a considerable period • No residue after pregnancy Early Pregnancy detection
  8. 8. Accurate method of pregnancy diagnosis only from day 30-45 post mating Risk of iatrogenic embryonic mortality No information about the viability of the embryo/fetus during stages of pregnancy Compared to cattle, rectal palpation in buffaloes must be gentle as the rectal mucosa is more fragile and bleed easily Transrectal Palpation
  9. 9. In cow/ buffalo can be adopted successfully from Day 28-30 after service At Day 30 it is possible to observed the fetal heart beats The sensitivity reaching 100% from Day 31 after mating onward Main advantage-can give an accurate diagnosis It is necessary to have a proficient operator Transrectal ultrasonography
  10. 10. This test is based on the milk progesterone concentration 21-24 days post breeding. The milk progesterone parallels blood progesterone. It is important to know that this can only be used with known AI or breeding dates and the test cannot be done randomly in a herd. It is based on the fact that if progesterone is low 21 days post-breeding then there is no chance that the cow can be pregnant. Remember that a cow must have progesterone if pregnant Therefore the test is 100% accurate at non- pregnancy diagnosis when the progesterone is low. Milk progesterone assay
  11. 11. If Progesterone is high 21 days after breeding the cow 'should' be pregnant
  12. 12. ∗ If Progesterone is low 21 days after breeding (or anytime) the cow cannot be pregnant ∗ However, if the progesterone is high, the test is only 80 % accurate at diagnosing cows pregnant…..because there may be mistakes and cows may be bred that are not actually in heat……. ∗ These cows would therefore have high progesterone 21 days later, yet not be pregnant ∗ Also cows with short and long cycles can cause misinterpretation of the test
  13. 13. ∗ A false positive if breeding was not done at estrus ∗ The progesterone 21 days later may be high and indicate a pregnant cow ∗ The blue line indicates the 2 ng/ml threshold value for positive ∗ These cows would therefore have high progesterone 21 days later, yet not be pregnant ∗ Also cows with short and long cycles can cause misinterpretation of the test
  14. 14. ∗ Trend in serum progesterone (P4) related to pregnancy outcome in buffalo cows artificially inseminated (day 0=AI). (Barbato and Barile, 2012)
  15. 15. Oestrous Cycle in Buffalo (Source: FAO)
  16. 16. Perioestrus endocrine changes in the buffalo cow (Seren et al., 1994)
  17. 17. ∗ The Early Pregnancy Factor (EPF) or Early Conception Factor (ECF) test was reported accurate on day 2 but the test was later shown to be inaccurate at any time. Other tests measure the biological response to interferon-t (day 16 to 18), progesterone in blood or milk (day 18 to 23), or pregnancy associated glycoproteins (PAG; after day 25) Some candidate molecules associated with early pregnancy in cattle (Lucy et al., 2011)
  18. 18. This protein molecule was first identified in pregnant mice (Morton et al., 1987) and later in sheep and cattle (Nancarrow et al., 1981) by using the rosette inhibition bioassay With this assay, EPF was detected in the serum of all mammals tested within 24 to 48 h of fertilization and disappeared within 24 to 48 h after death or removal of embryo (Morton et al.,1987) Early Conception Factor (ECF)
  19. 19. A chemical test for pregnancy termed Early Pregnancy Factor (EPF) or Early Conception Factor (ECF) was proposed and marketed in the 1990’s (Concepto Diagnostics, Knoxville,TN) This molecule was supposedly present in the blood of pregnant cattle within two days after conception The exact nature of this molecule (an immunosuppressive glycoprotein protein) and how it got into circulation were not well defined A kit for pregnancy diagnosis reached the market but three different studies found that the kit was unreliable for pregnancy diagnosis (Cordoba et al., 2001; Gandy et al., 2001; Ambrose et al., 2007)
  20. 20. Pregnancy-associated glycoprotein (also called pregnancy-specific protein B, pregnancy specific protein 60 and SBU-3 antigen) constitute a large family of glycoproteins expressed in the outer epithelial cell layer of the placenta of Eutherian species They are synthesized by mono and binucleate trophoblastic cells, some of them being secreted in maternal blood from the moment when the conceptus becomes more closely attached to the uterine wall and formation of placentomes begins Pregnancy-associated Glycoproteins
  21. 21. ∗ A 14 weeks buffalo fetus with visible placentomes (button- shaped structures) characteristic of ruminant placentation
  22. 22. The PAG (Pregnancy-associated glycoprotein) family were isolated from cotyledons of cow, ewe, goat, buffalo, bison, moose and elk PAG belong to the aspartic proteinase gene family However, most PAG molecules are assumed to be enzymatically inactive due to key mutations within their binding cleft Cattle, sheep and probably all ruminants possess many, possibly 100 or more, PAG genes
  23. 23. In bovine, 22 boPAG genes (boPAG-1 to boPAG-22) were cloned and fully sequenced The number of PAG gene is lower in ovine (15 genes) and caprine species (about 11 genes) Several bovine, ovine, caprine and buffalo closely related PAG molecules (63–87% N terminal amino acid identities) have been made available and have been used to produce antisera for radioimmunoassay (RIA) development
  24. 24. 1. BioPRYN BioTracking, LLC, Moscow, ID http://www.biotracking.com/ 2. DG29 Conception Animal Reproduction Technologies, Beaumont, QC http://www.conception-animal.com/test_an.html 3. IDEXX Bovine Pregnancy Test IDEXX Laboratories, Inc., Westbrook, ME, http://www.idexx.com/view/xhtml/en_us/livestock-poultry/ruminant/lpd-bovine- pregnancy-test.jsf ∗None of these tests are cow-side or on-farm, so blood samples must be collected by farm personnel and sent by courier to a local or regional laboratory that runs the assay. Results are then returned to the farm via email, usually within 24 to 72 h. Currently, three non-pregnancy tests based on detection of PAGs in maternal serum are commercially available for use by dairy farmers
  25. 25. Bovine interferon tau (boIFN-t) is one of the principal proteins secreted by the bovine conceptus from 16 to 25 days It has been showed that the “embryonic signals” emitted by the conceptus of the ruminants, initially called trophoblastin or trophoblastic protein, constitute actually the same subclass of interferon, pertaining to the family of interferon alpha In the ewe, the interferon tau (ovIFN-t) is produced between 13th to 21st days of gestation Interferon tau (IFN-t) regulates uterine Mx gene expression in ewes; however, the only known role for Mx gene is in the immune response to viral infection. Interferon Tau
  26. 26. It is hypothesized that Mx gene functions as a conceptus- induced component of the antiluteolytic mechanism and/or regulator of endometrial secretion or uterine remodeling during early pregnancy Molecules belonging to the subclass of IFN-t were also identified on the level of the trophoblastic cells of the caprine conceptus from 14 to 17 days. However from the 18th day, when the establishment starts, the caprine IFN-t is not detected any more, suggesting that in this species, other factors are necessary from the 18th day of gestation to provide the luteotropic function These effects range from pregnancy recognition signaling in ruminants through IFN tau to effects on cellular functions of the uterus and uterine vasculature
  27. 27. CNAs (Circulating Nucleic Acids) can also be used as markers for detection of early pregnancy in cattle (Mayer et al., 2012)
  28. 28. Relaxin can be determined in the peripheral circulation of pregnant bitches at 20-30 days of gestation, whereas it is absent in non-pregnant bitches at all stages of the reproductive cycle This relaxin is known to be produced by the placenta in the bitch and cat and is thought to contribute to its maintenance by inhibiting uterine activity In the domestic dog it has been established as a pregnancy-specific hormone Relaxin assay…..not for bovines/buffaloes ?
  29. 29. Ideal pregnancy biomarker characteristics Specifically up-regulated or down-regulated during pregnancy Expressed over a considerable period of time to give ample time for diagnosis Present in easily accessible body fluids like serum, milk, urine, vaginal discharges etc Preferably to have the ability to reflect age as well as viability of the conceptus Least affected by non-animal factors like feed, environment and drug interactions Testing method to have the ability to reveal the result immediately
  30. 30. None of the present day methods qualifies as an ideal diagnostic due to limitations of accuracy, later stages of applicability as well as the requirement for elaborate instrumentation and laboratory set-up Further investigations on developing novel early pregnancy diagnostics for livestock species, especially buffaloes, are in progress and hopefully, very soon we should have a suitable marker / diagnostic test for early pregnancy in livestock species including buffalo
  31. 31. Roadmap through Integrated Genomics…….
  32. 32. A. MCQ Type 1. Most reliable indication of successful pregnancy in buffaloes occurs at __________ post insemination (a) Day 15 + (b) Day 23 + (c) Day 30 + (d) Day 40+ 2. if progesterone is low 21 days post-breeding then (a) There is good chance of cow being pregnant (b) There is no chance that the cow can be pregnant (c) The corpus luteum is fully formed (d) None 3. Which of the followings is not a biomarker associated with early pregnancy in cattle (a) EPF (b) insulin (c) PAG (d) IFN-tau Questions
  33. 33. 4. Which of the followings regarding ECF is not true? (a)It is a nucleic acid molecule (b)It was first identified in pregnant mice (c)It was identified by using the rosette inhibition bioassay (d)It was detected in the serum of all mammals tested within 24 to 48 h of fertilization 5. Which of the followings are true for PAGs (a)These constitute a large family of glycoproteins (b)These belong to the aspartic proteinase gene family (c)Most PAG molecules are assumed to be enzymatically inactive due to key mutations within their binding cleft (d)B and C only (e)All of the above
  34. 34. 6. Which of the following statements is false: (a)Bovine interferon tau (boIFN-t) is secreted by the bovine conceptus from 16 to 25 days (b)Interferon tau (IFN-t) regulates uterine Mx gene (c)CNAs (Circulating Nucleic Acids) can also be used as markers for detection of early pregnancy in cattle (d)Relaxin is present in non-pregnant bitches and absent in pregnant bitches at 20-30 days of gestation B. Q. Answer the followings in maximum 2-3 sentences only. (1)Early Pregnancy Biomarker (2)Progesterone for pregnancy diagnosis (3)Relaxin assay (4)Mx Gene (5)Name 3 Candidate molecules for early detection of pregnancy (6) Early pregnancy diagnosis (7)Name three non-pregnancy tests based on detection of PAGs in maternal serum
  35. 35. C. Q. Short Notes (8 lines) ∗ (1) Ideal Pregnancy biomarker characteristics ∗ (2) Why early pregnancy biomarkers are required? ∗ (3) ECF ∗ (4) Interferon-tau ∗ (5) Pregnancy associated glycoproteins D. Q. Long notes (3-4 pages) 1. Discuss in detail about the early pregnancy biomarkers for early pregnancy diagnosis in livestock with reference to cattle and buffaloes.
  36. 36. E. Write True/ False for the given statements: 1.Per-rectal palpation and Ultrasonography are indirect method of pregnancy diagnosis (Answer: False) 2.If Progesterone is high 21 days after breeding the cow 'should' be pregnant (Answer: True) 3.If Progesterone is low 21 days after breeding (or anytime) the cow cannot be pregnant (Answer: True) 4.Transrectal palpation is an accurate method of pregnancy diagnosis only from day 30-45 post mating (Answer: True) 5.In the domestic dog relaxin has been established as a pregnancy- specific hormone (Answer: True)

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