Correlation analysis of serum reproductive hormones and metabolites during multiple ovulation in sheep

Abstract

Background: The establishment of non-invasive diagnostic method for multiple ovulation prediction is helpful to improve the efficiency of multiple ovulation. The blood hormones and metabolites would be suitable indexes for this subject.

Methods: In this study, 86 estrus ewes (65 of induced estrus (IE) and 21 of spontaneous estrus (SE)) were selected and the blood samples were collected at the day before follicle-stimulating hormone (FSH) injection (1^st) and before artificial insemination (2^nd). The serum reproductive hormones ofFSH, luteinizing hormone (LH), 17β-Estradiol (E2), progesterone (P4) and anti-Mullerian hormone (AMH) were measured through enzyme linked immunosorbent assay (ELISA) and the untargeted metabolomics analysis was processed through LC-MS/MS. The embryos were collected after 6.5 days of artificial insemination.

Results: In total, 975 and 406 embryos were collected in IE and SE group, respectively. The analysis of reproductive hormones showed that concentrations of FSH, E2 and AMH were positive correlated with the embryo yield while concentrations of LH and P4 were negative correlated in both group at 1^st detection. At 2^nd detection, the trends of reproductive hormones were similar with 1^st except P4, which was positive correlated with embryo yield. The metabolomics analysis showed that 1158 metabolites (721 in positive iron mode and 437 in negative iron mode) were detected and 617 were annotated. In 1^st comparation of high and low embryonic yield populations, 56 and 53 differential metabolites were identified in IE and SE group, respectively. The phosphatidyl choline (PC) (19:0/20:5) and PC (18:2/18:3) were shared in two groups. In 2^nd comparation, 48 and 49 differential metabolites were identified in IE and SE group, respectively. The PC (18:1/18:2) and pentadecanoic acid were shared. Most differential metabolites were significantly enriched in amino acid, fatty acid metabolism, digestive system secretion and ovarian steroidogenesis pathways.

Conclusions: This study showed that FSH, P4, AMH, the PC relevant metabolites and some anomic acids could be potential biomarkers for embryonic yield prediction in ovine multiple ovulation. The results would help to explain the relation between blood material and ovarian function and provide a theoretical basis for the multiple ovulation prediction.

Keywords: Blood metabolome; Blood reproductive hormone; Multiple Ovulation; Sheep.

Fig. 1

Schedule of ovine multiple ovulation. At the start of experiment, the estrus was detected and the ewes were divided into CIDR induced estrus (IE) and Spontaneous estrus (SE) group (65 and 21, respectively). The IE group was treated as upper: the CIDR was inserted into vagina of non-estrus ewes at random day (D0); at D10 to 12, the FSH was injected for 6 times; at first time of FSH injection, the PMSG was injected synchronously; at fifth time of FSH injection, the PG was injected synchronously; the CIDR was removed at last time of FSH injection; at D13, the estrus was detected and AI was performed twice (12 h interval); the LH was injected at first AI; at D19, the embryos were collected via surgical uterus flushing. The SE group was treated as under: the estrus day of ewes as D0; at D13 to 15, the FSH was injected for 6 times; at first time of FSH injection, at last time of FSH injection, the PG was injected synchronously; at D16, the estrus was detected and AI was performed twice (12 h interval); the LH was injected at first AI; at D22, the embryos were collected via surgical uterus flushing

Fig. 2

The first time (1^st) Metabolomic analysis of serum from ewes with multiple ovulation treatment. PCA analysis results of IE group under positive ion mode (a) and negative ion mode (b) and SE group under positive ion mode (c) and negative ion mode (d). PLS-DA analysis results of IE group under positive ion mode (c) and negative ion mode (d) and SE group under positive ion mode (g) and negative ion mode (h)

Fig. 3

The second time (2^nd) Metabolomic analysis of serum from ewes with multiple ovulation treatment. PCA analysis results of IE group under positive ion mode (a) and negative ion mode (b) and SE group under positive ion mode (c) and negative ion mode (d). PLS-DA analysis results of IE group under positive ion mode (c) and negative ion mode (d) and SE group under positive ion mode (g) and negative ion mode (h)

Fig. 4

Volcano plot of first comparation analysis (1^st) between high and low embryonic yield populations of serum from ewes with multiple ovulation treatment. IE group under positive ion mode (a) and negative ion mode (b) and SE group under positive ion mode (c) and negative ion mode (d)

Fig. 5

Volcano plot of second comparation analysis (2^nd) between high and low embryonic yield populations of serum from ewes with multiple ovulation treatment. IE group of positive ion mode (a) and negative ion mode (b) and SE group of positive ion mode (c) and negative ion mode (d)