Pregnancy-Associated Glycoprotein Level in Early Pregnancy Detection with Prediction of Fetal Number and Gender in Iraqi Local Ewes
Article Sidebar
Main Article Content
Abstract
Early pregnancy detection is an important tool in successful reproductive management in ewes. The objective of this study was to estimate the concentration of serum pregnancy-associated glycoprotein (PAG) during gestation using sheep ELISA kit in order to early distinguish between pregnant and non-pregnant ewes and the possible association with litter size, and fetal gender. Estrus was synchronized and blood samples were collected at day 0 (day of mating), 18, 30, 60, and 120 after mating from 18 Iraqi cross breed synchronized ewes. The result of PAG level (ng/ml) during estrus has shown insignificant difference between G1(CIDR group), G2(vaginal sponge group) and G3 (control group), while at day 18, 30, 60 and 120 of pregnancy was significantly (p˂0.05) higher in G1 and G2, in comparison with G3. The discrimination value (cutoff point) for detection of pregnancy by PAG test was ˃ 0.915 ng/ ml (using ROC Curve) and based on breeding data from day 18 PAG values were detectable in all pregnant ewes. It was also proved that mean values were significantly (P˂0.05) higher in twin than single pregnancies and significantly (P˂0.05) higher in male single than female single pregnancy in crossbred Iraqi ewes according to the data collected from day 0 to the end of pregnancy and follow the pregnancy up to the day of birth. Altogether, it is concluded that, ovine pregnancy may be reliably detected with fetal number and gender from day 18 after mating onward by using sheep ELISA kit of pregnancy associated glycoprotein.
Received: 14 May 2024
Revised: 29 June 2024
Accepted: 18 August 2024
Published: 28 December 2024
Downloads
Article Details
How to Cite
References
Commun L, Velek K, Barbry JB, Pun S, Rice A, Mestek A, et al. Detection of pregnancy-associated glycoproteins in milk and blood as a test for early pregnancy in dairy cow. J Vet Diagn Invest. 2016;28(3):207-213. https://doi.org/10.1177/1040638716632815
2. Kaya MS, KÖSE M, Bozkaya F, Mutlu H, Uçar EH Atli MO. Early pregnancy diagnosis using a commercial ELISA test based on pregnancy-associated glycoproteins in Holstein-Friesian heifers and lactating cows. Turk J Vet Anim Sci. 2016;40:694-699.
https://doi.org/10.3906/vet-1602-41
3. González-Bulnes A, Pallares P, Vazquez MI. Ultrasonographic imaging in small ruminant reproduction. Reprod Domest Anim. 2010; 45(2):9-20. https://doi.org/10.1111/j.1439-0531.2010.01640.x
4. Karen A, Beckers JF, Sulon J, de Sousa NM, Szabados K, Reczigel J, et al. Early pregnancy diagnosis in sheep by progesterone and pregnancy associated glycoprotein tests. Theriogenology. 2003;59:1941-8. https://doi.org/10.1016/S0093-691X(02)01289-X
5. de Miranda ESCC, Dias da Costa RL, Roncato Duarte KM, Machado DC, Paro de Paz CC, Beltrame RT. Visual ELISA for detection of pregnancy-associated glycoproteins (PAGs) in ewe serum. Theriogenology. 2017;97:78-82. https://doi.org/10.1016/j.theriogenology.2017.04.026
6. Barbato O, Sousa NM, Debenedetti A, Canali C, Todini L, Beckers JF. Validation of a new pregnancy-associated glycoprotein radioimmunoassay method for the detection of early pregnancy in ewes. Theriogenology. 2009;72:993-1000.
https://doi.org/10.1016/j.theriogenology.2009.06.020
7. Karen A, Sousa NM, Beckers JF, Bajcsy AC, Tibold J, Mádl I, et al. Comparison of a commercial bovine pregnancyassociated glycoprotein ELISA test and a pregnancy-associated glycoprotein radioimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim Reprod Sci. 2015;159:31-37. https://doi.org/10.1016/j.anireprosci.2015.05.005
8. Rovani MT, Skrebsky Cezar A, Lazzari Rigo M, Garziera Gasperin B, da Nobrega JE, Dias Torres F, et al. Evaluation of a bovine pregnancy glycoprotein enzyme-linked immunosorbent assay kit for serological diagnosis of pregnancy in sheep. Cienc Rural. 2016;46:362-367. https://doi.org/10.1590/0103-8478cr20150270
9. Alabart JL, Lahoz B, Folch J, Martí JI, Sánchez P, Delahaut P, et al. Early pregnancy diagnosis in sheep by plasmatic pregnancy associated glycoprotein (PAG) enzyme immune-assay (EIA) kit. XXXV Congreso de la SEOC. Valladolid, Spain Instituto Tecnológico Agrario, Junta de Castilla y Leó. 2010; p. 199-202.
10. Zoli AP, Beckers JF, Wouters-Ballman P, Closset J, Falmagne P, Ectors F. Purification and characterization of a bovine pregnancyassociated glycoprotein. Biol Reprod. 1991;45:1-10. https://doi.org/10.1095/biolreprod45.1.1
11. Garbayo JM, Green JA, Mannikam M, Beckers JF, Kiesling DO, Early AD, Roberts M. Caprine pregnancy associated glycoproteins (PAG): their cloning, expression, and evolutionary relationship to other PAG. Mol Reprod Dev. 2000;57:311-322.
https://doi.org/10.1002/1098-2795(200012)57:4<311::AID-MRD2>3.0.CO;2-F
12. Hashizume K, Ushizawa K, Patel OV, Kizaki K, Imai K, Yamada O, et al. Gene expression and maintenance of pregnancy in bovine roles of trophoblastic binucleate cell specific molecules. Reprod Fert Dev. 2007;19:79-90. https://doi.org/10.1071/RD06118
13. Sousa NM, Ayad A, Beckers JF, Gajewski Z. Pregnancy associated glycoproteins (PAG) as pregnancy markers in the ruminants. J Phy Pharm. 2006;57(8):153-171.
14. Redden R, Passavant C. Efficacy of pregnancy-specific protein B assay to detect pregnancy and lambing rates in sheep. Sheep Goat Res J. 2013;28:21-24.
15. Mercadante PM, Waters KM, Mercadante VR, Lamb GC, Elzo MA, Johnson SE, et al. Subspecies differences in early fetal development and plasma pregnancy-associated glycoprotein concentrations in cattle. J Anim Sci. 2013;91:3693-701. https://doi.org/10.2527/jas.2012-6130
16. Serrano B, Lopez-Gatius F, Santolaria P, Almeria S, Garcia-Ispierto I, Bech-Sabat G, et al. Factors affecting plasma pregnancy-associated glycoprotein 1 concentrations throughout gestation in highproducing dairy cows. Reprod Domest Anim. 2009;44:600-605.
https://doi.org/10.1111/j.1439-0531.2007.01025.x
17. Lobago F, Bekana M, Gustafsson H, Beckers JF, Yohannes G, Aster Y, et al. Serum profiles of pregnancy-associated glycoprotein, oestrone sulphate and progesterone during gestation and some factors influencing the profiles in Ethiopian Borana and crossbred cattle. Reprod Domest Anim. 2009;44:685-92. https://doi.org/10.1111/j.1439-0531.2007.01049.x
18. Thompson IM, Tao S, Branen J, Ealy AD, Dahl GE. Environmental regulation of pregnancy-specific protein B concentrations during late pregnancy in dairy cattle. J Anim Sci. 2013;91:168-73. https://doi.org/10.2527/jas.2012-5730
19. Ward JW, Wooding FB, Fowden AL. The effects of cortisol on the binucleate cell population in the ovine placenta during late gestation. Placenta. 2002;23:451-458. https://doi.org/10.1053/plac.2002.0834
20. Haugejorden G, Waage S, Dahl E, Karlberg K, Beckers JF, Ropstad E. Pregnancy associated glycoproteins (PAG) in postpartum cows, ewes, goats, and their offspring. theriogenology. 2006;66:1976-84. https://doi.org/10.1016/j.theriogenology.2006.05.016
21. El Amiri B, Sousa NM, Alvarez Oxiley A, Hadarbach D, Beckers JF. Pregnancy-Associated glycoprotein (PAG) concentration in plasma and milk samples for early pregnancy diagnosis in Lacaune dairy sheep. Res Vet Sci. 2015;99:30-36.
https://doi.org/10.1016/j.rvsc.2014.12.016
22. El Amiri B, Sulon J, Karen A, Alvarez AV, Cogniè Y, Sousa NM, et al. Measurement of ovine pregnancy-associated glycoprotein (PAG) during early pregnancy in Lacaune sheep. Reprod Domest Anim. 2007;42:257-262. https://doi.org/10.1111/j.1439-0531.2006.00761.x
23. De Carolis M., Barbato O. , Acuti G., Trabalza-Marinucci M., de Sousa N.M., Canali C, et al. Plasmatic Profile of Pregnancy-Associated Glycoprotein (PAG) during Gestation and Postpartum in Sarda and Lacaune Sheep Determined with Two Radioimmunoassay Systems. animals. 2020;10:1502. https://doi.org/10.3390/ani10091502
24. Steckeler P, Weber F, Zerbe H, Rieger A, Voigt K. Evaluation of a bovine visual pregnancy test for the detection of pregnancy-associated glycoproteins in sheep. Reprod Domest Anim. 2019;54:280-288. https://doi.org/10.1111/rda.13356
25. Beriot M, Tchimbou AF, Barbato O, Beckers JF, de Sousa NM. Identification of pregnancy-associated glycoproteins and alpha protein in fallow deer (Dama dama) placenta. Acta Vet. Scandinava. 2014;56(1):4. https://doi.org/10.1186/1751-0147-56-4
26. Barbato O, Menchetti L, Sousa NM, Malfatti A, Brecchia G, Canali C, Beckers JF, Barile VL. Pregnancy-Associated glycoproteins (PAGs) concentrations in water bu aloes (Bubalus bubalis) during gestation and the postpartum period. theriogenology. 2017;97:73-77.
https://doi.org/10.1016/j.theriogenology.2017.04.021
27. Karen A, Bajcsy AC, Minoia R, Kovács R, Sousa NM, Beckers JF, et al. Relationship of progesterone, bovine pregnancy-associated glycoprotein-1, and nitric oxide with late embryonic and early fetal mortalities in dairy cows. J Reprod Dev. 2014;60:162-167.
https://doi.org/10.1262/jrd.2013-033
28. Piechotta M, Bollwein J, Friedrich M, Heilkenbrinker T, Passavant C, Branen J, et al. Comparison of commercial ELISA blood test s for early pregnancy detection in dairy cows. J Reprod Dev. 2011;57:72-75. https://doi.org/10.1262/jrd.10-022T
29. Breukelman SP, Perényi Z, Taverne MAM, Jonker H, Van der Weijden GC, Vos PLAM, et al. Characterisation of pregnancy losses after embryo transfer by measuring plasma progesterone and bovine pregnancy-associated glycoprotein-1 concentrations. Vet J. 2012;194:71-76. https://doi.org/10.1016/j.tvjl.2012.02.020
30. Engelke J, Knaack H, Linden M, Feldmann M, Gundling N, Gundelach Y, et al. Identification of embryonic/fetal mortality in cows by semiquantitative detection of pregnancy-associated glycoproteins. Livest Sci. 2015;178:363-370. https://doi.org/10.1016/j.livsci.2015.05.030
31. Pohler KG, Pereira MH, Lopes FR, Lawrence JC, Keisler DH, Smith MF, et al. Circulating concentrations of bovine pregnancy-associated glycoproteins and late embryonic mortality in lactating dairy herds. J Dairy Sci. 2016;99(2):1584-1594.
https://doi.org/10.3168/jds.2015-10192
32. Younis LS, Hatif SA. AA-NAT melatonin gene polymorphism in ewes lambing out of season. Onl J Vet Res. 2017;21(3):118-125.
33. Ajafar MH, Kadhim AH, AL-Thuwaini TM. The reproductive traits of sheep and their influencing factors. Reviews in Agric Sci. 2022;10:82-89. https://doi.org/10.7831/ras.10.0_82
34. Hatif SA, Younis LS. Nocturnal and diurnal plasma melatonin in season and non-seasonal sheep during spring. Onl J Vet Res. 2018a;22(6):513-517.
35. Hatif SA, Younis LS. Effect of aryl alkyl amine-N-acetyl-transferase gene polymorphism on melatonin in non-seasonal ewes. Onl J Vet Res. 2018b;22(5):356-361.
36. Al-Hamedawi TM, Khammas DJ, Mohammed AH. Effect of using various doses of Bromocriptine in estrus induction and subsequent fertility in lactating anestrus Iraqi Ewes. Iraqi J Vet Med. 2016;40(2):14-19. https://doi.org/10.30539/iraqijvm.v40i2.104
37. Al-Hamedawi TM, Mohammed AH, Al-Yasiri EA, Athab ML. Comparative study between melatonin hormone and vaginal sponges plus eCG on effect of fertility in lactating Iraqi Nuaimy ewes. Plant Archives. 2020; 14(93.3): 4-82.
38. Abas HKEAA, Al-Hamedawi TM, Ajeel HM. Pregnancy Rate in Synchronized Iraqi Awassi Ewes Inseminated Artificially and Naturally. Indian J Forensic Med Toxicol. 2022;16(1):1669-1673. https://doi.org/10.37506/ijfmt.v16i1.17808
39. Kadhim Q, Hussain SO. Assessing the Influence of Administration of Kisspeptin-10 on LH Release and Reproductive performance in estrus synchronized ewes. Egypt J Vet Sci. 2024a; 55(2):499-505. https://doi.org/10.21608/ejvs.2023.239749.1632
40. Kadhim QJ, Hussain SO. Determination the serum oestrogen Concentration and Alkaline phosphatase activity during pregnancy and parturition in oestrus synchronized goat. Iraqi J Vet Med. 2014;38(2):95-99. https://doi.org/10.30539/iraqijvm.v38i2.229
41. Hussein KA, Omran SN, AL-Rawi HM. The Use of Ultrasonography to Detect the Response of Local Awassi Ewes to Synchronization of Estrous and Superovulation to Different Intravaginal Progestagen sponge and eCG. Iraqi J Vet Med. 2007;31(1):41-49.
https://doi.org/10.30539/iraqijvm.v31i1.802
42. Hameed N, Khan MIUR, Zubair M, Andrabi SMH. Approaches of estrous synchronization in sheep: Developments during the last two decades: A review. Trop Anim Health Prod. 2021;53(5):485. https://doi.org/10.1007/s11250-021-02932-8
43. Abdulkareem TA, Muhammad SJ, Yousif AN. Effect of kispeptin-10 as an alternative to eCG in estrus synchronization protocol on improving the reproductive performance of karadi ewes. Iraqi J Agric Sci. 2021;52(3):535-546. https://doi.org/10.36103/ijas.v52i3.1340
44. Kadhim Q, Hussain SO. The Impact of Administering Kisspeptin-10 Peripherally on FSH Release and Reproductive Performance in Estrus Synchronized Ewes. Indian Vet J. 2024b;101(2):15-22. https://doi.org/10.62757/IVA.2024.101.2.15-22
45. Abood HK. Clinical study of experimentally induced vitamin E and selenium deficiency in Awassi ewes and their newborn lambs: HK Abood, AMH Judi and AA AL-Ani. Iraqi J Vet Med. 2012;36(2):158-162. https://doi.org/10.30539/iraqijvm.v36i2.458
46. Abdulkareem TA, Eidan SM, Al-Maliki LA, Al-Saidy FK, Mahdi MR. Reproductive performance of Iraqi Awassi ewes owned by sheep owners and extension farms in response to flushing and estrus synchronization regimes. Iraqi J Agric Sci. 2014;45:328-334.
47. Hussain SO, Hussain KA, Al-Ani AA. Study on post-partum uterine involution by Ultrasonography and progesterone profile in local goats in Iraq. Iraqi J Vet Med. 2016;40(1):151-156. https://doi.org/10.30539/iraqijvm.v40i1.153
48. Alrawy IY, Hussain SO. Efficacy of Gonadotropin Releasing Hormone on Puberty in Iraqi Awassi Cross Breed Ewe Lambs. Egypt J Vet Sci. 2025;56(1):61-68. https://doi.org/10.21608/ejvs.2024.262822.1782
49. Hussain SO, Badry KA, Zalzala SJ, Zakri AM. Activity of transaminase enzyme and testosterone hormone in blood of Awassi rams during different season. Asian Pac J Reprod. 2017;6(5):217-220. https://doi.org/10.4103/2305-0500.215932
50. SAS. Statistical Analysis System, User's Guide. Statistical. Version 9.6th ed. SAS. 2018;Inst. Inc. Cary. N.C. USA.
51. MedCalc Statistical Software version 16.4.3 (MedCalc Software bvba, Ostend, Belgium; 2016; https://www.medcalc.org)"
52. Al-Rawi MAO, Hussain SO. Impact of CIDR-PMSG and MAP sponges- PMSG treatment of reproductive performance of ewes with assessment of progesterone concentration during treatment and pregnancy. O&G Forum. 2024;34(3):1220-1240.
53. Khan D, Khan H, Ahmad N, Tunio MT, Tahir M, Khan MS, Khan RU. Early pregnancy diagnosis using pregnancy-associated glycoproteins in the serum of pregnant ruminants. Pakistan J Zool. 2020;52(2):785-788.
https://doi.org/10.17582/journal.pjz/20190527080541
54. Silva E, Sterry RA, Kolb D, Mathialagan N, McGrath MF, Ballam JM. Accuracy of a Pregnancy-Associated Glycoprotein ELISA to Determine Pregnancy Status of Lactating Dairy Cows Twenty-Seven Days After Timed Artificial Insemination. J Dairy Sci. 2007;90:4612-4622. https://doi.org/10.3168/jds.2007-0276
55. García-Ispierto I, Almería S, Serrano B, de Sousa NM, Beckers JF, López Gatius F. Plasma Concentrations of Pregnancy-Associated Glycoproteins Measured Using Anti-Bovine PAG-2 Antibodies on Day 120 of Gestation Predict Abortion in Dairy Cows Naturally Infected with Neospora caninum. Reprod Dom Anim. 2013;48(4):613-618. https://doi.org/10.1111/rda.12134
56. Wallace JM, Da Silva P, Aitken RP, Cruickshank MA. Maternal endocrine status in relation to pregnancy outcome in rapidly growing adolescent sheep. J Endocrinol. 1997;155(2):359-368. https://doi.org/10.1677/joe.0.1550359
57. Ranilla MJ, Sulon J, Carro MD, Mantecon AR, Beckers JF. Plasmatic profiles of pregnancy-associated glycoprotein and progesterone levels during gestation in Churra and Merino sheep. theriogenology. 1994;42(3):537-545. https://doi.org/10.1016/0093-691X(94)90691-B
58. Lawson BC, Shahzad AH, Dolecheck KA, Martel EL, Velek KA, Ray DL, et al. A pregnancy detection assay using milk samples: evaluation and considerations. J Dairy Sci. 2014;97(10):6316-6325. https://doi.org/10.3168/jds.2014-8233
59. López-Gatius F, Garbayo JM, Santolaria P, Yániz J, Ayad A, de Sousa NM, et al. Milk production correlates negatively with plasma levels of pregnancyassociated glycoprotein (PAG) during the early fetal period in high producing dairy cows with live fetuses. Domest Anim Endocrinol. 2007;32(1):29-42. https://doi.org/10.1016/j.domaniend.2005.12.007
60. Green JA, Xie S, Quan X, Bao B, Gan X, Mathialagan N, et al. Pregnancyassociated glycoproteins exhibit spatially and temporally distinct expression patterns during pregnancy. Biol Reprod. 2000;62(6):1624-1631. https://doi.org/10.1095/biolreprod62.6.1624
61. Kaulfuß KH, Schram D, Berttram M. Effect of genotype, age of dams, litter size, birthweight, and rams on morphological parameters of the placenta in sheep. Dtsch Tierarztl Wochenschr. 2000;107(7):269-275.
62. Ranilla MJ, Sulon J, Mantecon AR, Beckers JF, Carro MF. Plasma pregnancy-associated glycoprotein and progesterone concentrations in pregnant Assaf ewes carrying single and twin lambs. Small Rumin Res. 1997;24:125-131. https://doi.org/10.1016/S0921-4488(96)00922-4
63. Singh SP, Ramachandrana N, Sharmaa N, Goela AK, Gururajb K, Kharchea SD. Temporal changes in plasma profile of pregnancy-associated glycoprotein, progesterone and estrone sulfate associated with fetal number during early- and mid-pregnancy in goats. Anim Reprod Sci. 2019;205:115-125.https://doi.org/10.1016/j.anireprosci.2019.04.010
64. Serrano-Pérez B, Garcia-Ispierto I, Sousa NM, Beckers JF, Almeida S, López-Gatius F. Gamma interferon production and plasma concentrations of pregnancy-associated glycoproteins 1 and 2 in gestating dairy cows naturally infected with Neospora caninum. Reprod Dom Anim. 2014;49(2):275-280. https://doi.org/10.1111/rda.12267
65. Ledezma-Torres RA, Beckers JF, Holtz W. Assessment of plasma profile of pregnancy-associated glycoprotein (PAG) in sheep with a heterologous (anticaPAG55+ 59) RIA and its potential for diagnosing pregnancy. Theriogenology. 2006;66(4):906-912.
https://doi.org/10.1016/j.theriogenology.2006.02.031
66. Vandaele L, Verberckmoes S, El Amiri B, Sulon J, Duchateau L, Van Soom A, et al. Use of homolougous radioimmunoassay (RIA) to evaluate the effect of maternal and foetal parameters on pregnancy-associated glycoprotein (PAG) concentrations in sheep. Theriogenology. 2005;63(7):1914-1924. https://doi.org/10.1016/j.theriogenology.2004.08.009
67. Godfrey RW, Collins JR, Hensley EL, et al. Estrus synchronization and artificial insemination of hair sheep ewes in the tropics. Theriogenology 1999;51(5):985-97. https://doi.org/10.1016/S0093-691X(99)00044-8
68. Letelier CA, Contreras-Solis I, Garcı'a-Ferna'ndez RA, et al. Ovarian follicular dynamics and plasma steroid concentrations are not significantly different in ewes given intravaginal sponges containing either 20 or 40 mg of fluorogestone acetate. Theriogenology. 2009;71(4):676-82. https://doi.org/10.1016/j.theriogenology.2008.09.030
69. Kalyan De, Kumar D, Balaganur K, Gulyani R, Naqvi SMK. Effect of breeding season on fertility of sheep following estrus synchronization and fixed-time artificial insemination under field conditions in semi-arid tropical region, Biol. Rhythm Res. 2016. 10.1080/09291016.2016.1197497
70. Naderipour H, Yadi J, Shad A, Sirjani MA. The effects of three methods of synchronization on estrus induction and hormonal profile in Kalkuhi ewes: a comparison study. Afr J Biotechnol. 2012;11(2):530-533. https://doi.org/10.5897/AJB11.2718
71. Ozyurtlu N, Kucukaslan I, Cetin Y. Characterization of oestrous induction response, oestrous duration, fecundity, and fertility in Awassi ewes during the non-breeding season utilizing both CIDR and intravaginal sponge treatments. Reprod Dom Anim. 2010;45(3):464- 467. https://doi.org/10.1111/j.1439-0531.2008.01246.x
72. Yu XJ, Wang J, Bai YY. Estrous synchronization in ewes: The use of progestogens and prostaglandins, Acta Agriculturae Scandinavica, Section A-Anim Sci. 2019. https://doi.org/10.1080/09064702.2019.1674373