Development of a pregnancy associated glycoprotein assay to detect late embryonic mortality in cattle
Abstract
[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Embryonic mortality (EM) decreases profitability of a beef or dairy operation because of the impact that it is has on delaying the time of conception and consequently calving date. Most of the embryonic/fetal losses occur prior to maternal recognition of pregnancy, during the early implantation process, or during placentation (Vanroose et al., 2000). There are two classifications of embryonic mortality: early embryonic mortality (EEM), which occurs prior to day 28 following insemination, and late embryonic mortality (LEM), which occurs between days 28 and 42 with day 42 defined as the beginning of the fetal period (Karen et al., 2014; Whitlock and Maxwell, 2008). The establishment and maintenance of pregnancy is a complex physiological process. In cattle, fertilization rate is high (i.e. [about]90%); however, there is a 20 to 25% loss of embryos by the time of maternal recognition of pregnancy (day 15-16; day 0 = estrus; Humblot (2001). Furthermore, there is an additional loss of embryos (i.e. 4 to 40 %) that occurs after day 28 (LEM). In dairy cows, the highest incidence of LEM occurs from days 25 to 98 after induced ovulation and ranged from 14 % to 40 % (Cartmill et al., 2001a ; Perry et al., 2005; Vasconcelos et al., 1999). Although the percentage of LEM is smaller than EEM, it can cause serious economic losses due to a cow not conceiving during the breeding season or delayed conception (Silke et al., 2002). EM can also result in slower genetic progress and can negatively affect economic efficiency in the cattle industry (Dunne et al., 2000). Investigators have hypothesized that factors contributing to LEM may include inadequate oocyte competence (Lonergan et al., 2003), decreased luteal progesterone secretion (Rhinehart et al., 2009), inadequate uterine environment (Barnes, 2000), and(or) placental insufficiency (Facciotti et al., 2009). In order to investigate the possible etiological causes of LEM and to develop management strategies for decreasing this form of reproductive loss, the identification of a circulating marker of pregnancy that can be used to predict embryonic mortality/survival must be identified. Bovine pregnancy associated glycoproteins (PAGs) are members of a large gene family (>20 members). They are produced in the binucleate trophoblast cells of the placenta, they can be detected in the maternal circulation beginning around days 24 to 26 after insemination (Green et al., 2000; Pohler et al., 2013), and may serve as an indicator for placental development and function (Perry et. al., 2005). Pohler et al. (2013) reported that circulating concentrations of PAGs were higher on day 28 in cows that successfully maintained pregnancy until day 72 of gestation compared to cows that experienced LEM between days 27 to 72 of gestation. Although detection of PAGs has been reported to be an accurate method of diagnosing pregnancy, the efficacy of utilizing detection of PAGs, during early gestation (e.g. day 30), to predict LEM appears to depend on the assay platform (Pohler et al., 2013; Ricci et al., 2015). The ability of different PAG assays to accurately predict late embryonic mortality is likely influenced by the cross reactivity of specific antisera with different members of the PAG family. In many of the experiments described herein, circulating bovine PAGs were detected with different monoclonal antibodies (trapping antibody; A6, J2, L4, or a combination of all three [mix]) and a polyclonal antibody [second antibody; F2]) in a sandwich Enzyme Linked Immunosorbent Assay (ELISA) or with a commercial PAG ELISA pregnancy test. The long-term goal of this research is to identify specific antibodies directed against PAGs that can be used to accurately predict LEM in cattle between days 28 and 42 of gestation. The general hypothesis is that circulating concentrations of PAGs during early gestation will differ when measured with different antibody combinations and that specific antibody combinations in a sandwich ELISA will detect PAGs that are better predictors of LEM than other PAG members. The specific objectives of this study were as follows: 1) Determine if the pattern of secretion of circulating concentrations of PAGs in bovine serum on days 7, 24, 31, 45, 60, and 104 of gestation was different when using different monoclonal antibodies (trapping antibody; A6, J2, L4, or a combination of all three [mix]) with a polyclonal antibody [second antibody; F2]) in a sandwich ELISA and with a commercial PAG ELISA, 2) Determine if circulating concentrations of PAGs can be used to accurately determine if an animal is pregnant or not pregnant around day 30 of gestation when using individual monoclonal antibodies (A6, J2, L4, or a combination of all three [mix]) with a polyclonal antibody [F2]), 3) Determine if there is a specific monoclonal PAG antibody (A6, J2, L4, or a combination of all three [mix]) in conjunction with the F2 polyclonal second antibody that can serve as a more accurate predictor of LEM in cattle than the mix of monoclonal antibodies (mix) in combination with a different polyclonal antibody (45) and a commercial PAG ELISA, and 4) We examine the effect of ovulatory follicle size and circulating concentrations of postovulatory progesterone on circulating concentrations of PAGs on days 28 to 30 of gestation.
Degree
M.S.
Thesis Department
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