Identification of candidate blood biomarkers through metabolomics analysis in bovine superovulation

Abstract

Superovulation and embryo transfer technologies provide strong support for improving the productivity of cattle population. A non-invasive diagnostic method for superovulation prediction is necessary to improve its efficiency. Compared to macromolecular substances, there has been an increasing number of studies on small molecular metabolites as biomarkers. This study aimed to identify key biomarkers associated with superovulation outcomes in cows through serum metabolomics analysis. In this study, 36 induced estrus cows were selected, and the blood samples were collected at three time points: before FSH injection, before artificial insemination, and before embryo collection. Then, the cows were classified into high embryonic yield (HEY) and low embryonic yield (LEY) groups based on the total number of embryos. Furthermore, a serum untargeted metabolomics analysis of the two groups was conducted using liquid chromatography with tandem mass spectrometry (LC-MS/MS). A total of 372 embryos were collected. The metabolomics analysis revealed that 1,158 metabolites were detected, and 617 were annotated. In the before FSH injection samples, 121 differential metabolites were identified between the two groups. In the before artificial insemination samples, 129 differential metabolites were identified. In the before embryo collection samples, 201 differential metabolites were identified. A total of 11 differential metabolites were shared between the before FSH injection and before artificial insemination samples, while five differential metabolites were shared across all three samples. The majority of the differential metabolites were significantly enriched in pathways related to amino acid and fatty acid metabolism, digestive system secretion, and ovarian steroidogenesis. This study showed that phosphatidylcholine [PC; 14:0/22:1(13Z)], phosphatidylethanolamine [PE; DiMe (11, 3)], triacylglycerol [TG; 15:0/16:0/22:4 (7Z, 10Z, 13Z, 16Z)], phosphatidylinositol [PI; 16:0/22:2 (13Z, 16Z)], and phosphatidylserine [PS; 18:0/20:4(8Z, 11Z, 14Z, 17Z)] were differentially expressed in the serum during the superovulation period. These could serve as potential biomarkers for embryonic yield prediction in bovine superovulation. The lipid and amino acid metabolic pathways may have an impact on the ovarian response. The results of this study could provide novel screening indexes of donors for bovine superovulation, although the accuracy of the relevant factors requires further investigation.

Keywords: biomarker; cow; metabolome; serum; superovulation.

Figure 1

CIDR (Zoetis, New Zealand) was inserted into the vagina on day 0 (D0). Between days 9 and 12, a total of 500 μg of FSH (Stimufol^®, Belgium) was injected in eight doses, with each dose decreasing by 10%. On the fifth FSH injection (morning of D11), 300 μg of PG (Reprobiol, New Zealand) was injected synchronously. The CIDR was removed at the time of the final FSH injection. Estrus detection occurred on D13, and AI was performed twice (12 h interval). On D20, embryos were collected via uterus flushing after the cows were anesthetized with a caudal spinal injection of lidocaine hydrochloride.

Figure 2

Multivariate statistical analysis results of the metabolome. (A) PCA results. B–D: OPLS-DA results from three sampling time points: (B) before FSH injection; (C) before artificial insemination; and (D) before embryo collection. HEY1: High embryonic yield group at the sampling before FSH injection. LEY1: Low embryonic yield group at the sampling before FSH injection. HEY2: High embryonic yield group at the sampling before artificial insemination. LEY2: Low embryonic yield group at the sampling before artificial insemination. HEY3: High embryonic yield group at the sampling before embryo collection. LEY3: Low embryonic yield group at the sampling before embryo collection.

Figure 3

Differential metabolite analysis results. (A–C) Volcano plots for three sampling time points: (A) Before FSH injection; (B) before artificial insemination; and (C) before embryo collection. (D) Venn diagram of the differential metabolites across three sampling time points. HEY1: High embryonic yield group at the sampling before FSH injection. LEY1: Low embryonic yield group at the sampling before FSH injection. HEY2: High embryonic yield group at the sampling before artificial insemination. LEY2: Low embryonic yield group at the sampling before artificial insemination. HEY3: High embryonic yield group at the sampling before embryo collection. LEY3: Low embryonic yield group at the sampling before embryo collection.

Figure 4

KEGG enrichment results of differential metabolites. (A) Before FSH injection; (B) before artificial insemination; (C) before embryo collection.