Elsevier

Theriogenology

Volume 81, Issue 2, 15 January 2014, Pages 225-229
Theriogenology

Research article
Amino acid profiles in first trimester amniotic fluids of healthy bovine cloned pregnancies are similar to those of IVF pregnancies, but not nonviable cloned pregnancies

https://doi.org/10.1016/j.theriogenology.2013.09.012Get rights and content

Abstract

Somatic cell nuclear transfer (SCNT), or cloning, is one of the assisted reproductive technologies currently used in agriculture. Commercial applications of SCNT are presently limited to the production of animals of high genetic merit or the production of the most elite show cattle owing to its relatively low efficiency. In current practice, 20% to 40% of SCNT pregnancies do not result in viable offspring. In an effort to better understand some of the anomalies associated with SCNT pregnancies, we investigated amino acid compositions of first trimester amniotic fluid. In this retrospective study, amniotic fluids were collected from SCNT and control IVF pregnancies at Day 75 of gestation and grouped according to the pregnancy results: control IVF (IVF), viable SCNT pregnancies that resulted in live healthy calves (SCNT-HL), nonviable SCNT pregnancies that were aborted before Day 150 (SCNT-ED), and nonviable SCNT pregnancies that were aborted after Day 150 or produced deceased calves (SCNT-LD). High-performance liquid chromatography (HPLC) was used to analyze the concentrations of 22 amino acids (AAs) in the amniotic fluid samples. There were no differences in average AA concentrations between IVF and SCNT-HL groups, whereas SCNT-LD and SCNT-ED had higher levels of total AA concentrations. Concentrations of asparagine, citruline, arginine, and valine were significantly higher in the SCNT-LD group. Both SCNT-LD and SCNT-ED groups had relatively large intragroup variances in AA concentrations. Urea concentration was also measured in the SCNT amniotic fluid samples. No correlations between urea concentrations and arginine concentrations or pregnancy outcomes were found. The findings in this study not only deepen the understanding on SCNT pregnancy anomalies, but also provide a potentially useful screening tool for assessing viable and nonviable SCNT pregnancies.

Introduction

Suboptimal reproductive efficiency of somatic cell nuclear transfer (SCNT) impedes its broader application [1]. Although SCNT in vitro embryo development and pregnancy establishment rates are very similar to those of IVF embryos, the loss of SCNT pregnancies at later gestational stages is significantly higher, accounting for 20% to 40% of total embryos transferred [2]. Abnormal placentation is often observed in SCNT pregnancies, and is believed to be one of the major common defects identified in unsuccessful SCNT pregnancies [3], [4]. Aberrant molecular profiles in endometria and extra-embryonic tissues of SCNT transfers [5], [6] suggest that the placentation problem could begin as soon as embryos enter uteri. Changes in chemical compositions of amniotic fluid in SCNT pregnancies were also observed [7], indicating the alteration in the relative contribution of the fetal and placental tissues to the amniotic compartments. Thus, chemical compositions of amniotic fluid in as early as the first trimester could be potential markers for detecting early onset placentation defects and fetal anomalies.

Amniotic fluid provides an environment in which the fetus and its metabolic function develop. The volume and composition of amniotic fluid are therefore strictly regulated during development, reflecting the dynamic balance maintained through exchange between maternal circulation and fetal environment [8], [9], [10]. Amniotic fluid also provides the fetus with amino acids (AAs), which are the building blocks for biomolecules involved in physiologic growth and development, and precursors of nitrogenous substances with varied regulatory functions [11]. Any aberrations may elicit fetal stress or compromise fetal development. To the best of our knowledge, there has been no report on the study of AA profiles in amniotic fluids bearing SCNT fetuses. Thus, the objective of this study was to determine if the AA profiles in first trimester amniotic fluid reflected the competency of SCNT pregnancies.

In this study, we compared AA profiles of amniotic fluids from viable SCNT pregnancies that resulted in live healthy calves (SCNT-HL), nonviable SCNT pregnancies that were aborted or produced deceased calves (SCNT-ED), and IVF pregnancies with over 95% healthy calf delivery rate (SCNT-LD). Amniotic fluid was drawn at Day 75 of gestation. Day 75 of gestation was selected based on the practical consideration should AA profile be used as a screening marker for SCNT pregnancy competency. At this stage, the amniotic fluid volume is greatly increased and pregnancy termination can be performed if an AA profile indicates a nonviable pregnancy. Our results show that the AA profiles between viable SCNT pregnancies and IVF pregnancies are very similar; whereas, significant differences in the AA profiles exist between nonviable SCNT pregnancies and viable SCNT or IVF pregnancies.

Section snippets

Pregnancy preparation and amniotic fluid collection

We produced IVF and SCNT embryos using the protocols described previously [12] with the modification that synthetic oviductal fluid medium containing 0.4% BSA was used as an embryo culture medium. On Day 7, the embryos were examined morphologically and graded using the International Embryo Transfer Society grading system [13]. Stages 4 to 8 and grades 1 to 2 were transferred to synchronized Angus recipients. Pregnancies were confirmed by ultrasonography and pregnant recipients were monitored

Results

Average individual concentration of the majority of AAs and consequently the total AA concentration were elevated in nonviable SCNT pregnancies, compared with those in the viable SCNT and control IVF pregnancies (Table 1). Asparagine (Asn), citrulline (Cit), arginine (Arg), and valine (Val) were significantly higher (P < 0.03) in SCNT-LD compared with IVF and SCNT-HL. Concentrations of serine (Ser), glutamine (Glu), threonine (Thr), taurine (Tau), tyrosine (Tyr), isoleucine (Ile), and lysine

Discussion

Amino acids have important roles in embryonic survival, growth, and development [19]. Until now, little was known about changes in amniotic fluid AAs of compromised SCNT pregnancies. Our results show that AA concentration profiles are similar in IVF and viable SCNT pregnancies, whereas the concentrations are generally higher in amniotic fluids of nonviable SCNT pregnancies.

Amniotic fluid AAs mainly come from maternal and fetal circulation through the placenta during the first trimester [10],

Acknowledgments

The authors thank Dr. Thomas Bunch for helpful discussions and critical reading of the manuscript.

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    • Differences in amniotic amino acid concentrations between pregnancies obtained with transfer of vitrified thawed invitro-produced embryos and with natural mating in sheep

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      Weight at birth in this breed is quite constant [37], hence, it is highly unlikely that the use of different rams influenced fetal and placental development, and consequently amniotic AA concentration. Alanine, glycine, glutamic acid, and serine were the most abundant AAs in both experimental groups, in accordance with what was reported in naturally mated ewes [11] and in IVP bovine pregnancies [12]. Also, the dramatic twofold decrease in amniotic AA concentration observed from Days 60 to 80 of gestation is similar to what was previously reported in naturally mated ewes [11], but our data show a more pronounced, albeit not significant, decrease in V/T pregnancies compared with controls.

    1

    Present address: Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322-4815.

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