# Metabolomic Markers Reveal How hCG–Ketoprofen Intervention Increase Pregnancy Percentage Following Timed Artificial Insemination in Dairy Cows

## TL;DR

Combining hCG and ketoprofen improves pregnancy rates in dairy cows after artificial insemination by reducing inflammation and altering metabolism.

## Contribution

The novel finding is that hCG combined with ketoprofen increases pregnancy rates through metabolic and hormonal changes.

## Key findings

- Cows treated with hCG+ketoprofen had a 60.1% pregnancy rate, higher than the other groups.
- The treatment reduced oxidative stress and inflammation while altering lipid and amino acid metabolism.
- Metabolomic analysis showed upregulated tryptophan and serotonergic pathways and anti-inflammatory effects.

## Abstract

This study was conducted to evaluate whether combining human chorionic gonadotrophin (hCG) and ketoprofen can improve pregnancy percentages in dairy cows after timed artificial insemination (TAI). A total of 799 Holstein cows were assigned to three treatment groups: hCG-3, hCG-2 and hCG plus ketoprofen. The cows in the hCG plus ketoprofen group had the highest pregnancy percentage compared with those in the other two groups. The values obtained from metabolomic analysis of the serum collected on days 17 and 21 after TAI indicated reduced oxidative stress and inflammation, altered lipid and amino acid metabolism and hormonal differences in cows of the hCG+ketoprofen group compared with those of the hCG-3 group.

The objective of this study was to determine if the use of human chorionic gonadotrophin (hCG) and ketoprofen would affect the pregnancy percentages of dairy cows following timed artificial insemination (TAI). This experiment was conducted on a commercial dairy farm in China involving 799 healthy Holstein cows (2–4 parities) between October and December 2024. Cows were randomly assigned to three groups: hCG-3 = treated with (0 d: GnRH, 100 μg; 7–8 d: PGF2α, 0.4 mg; 52 h: GnRH, 100 μg; 14–16 h: TAI) + hCG 3 vials (300 µg)/cow on day 7 of TAI; hCG-2 group = TAI + 2 vials (200 µg) hCG/cow on day 7; and hCG+ketoprofen = TAI + 300 µg hCG/cow + ketoprofen 10 mL/cow on days 15 and 16 of TAI. Metabolomic profiling (untargeted and targeted) of 22 pregnant cows was conducted on serum collected on days 17 and 21 post-TAI. Results indicated greater pregnancy percentages in the cows of the hCG+ketoprofen-treated group compared to those in the other two groups (60.1% compared with 49.6% and 41.9%). The cows treated with hCG+ketoprofen had less oxidative stress markers, downregulation of arachidonic acid metabolism and upregulation of glycerophospholipid metabolism on day 17 after TAI, indicating that there was upregulation of tryptophan and serotonergic pathways, increased amino acid metabolism and continued anti-inflammatory effects on day 21 after TAI. These findings were confirmed by evaluation data collected by conducting the targeted metabolomic procedures, as indicated by the greater progesterone and melatonin and lesser 17-estradiol and 21-deoxycortisol concentrations. These findings indicate that combined hCG+ketoprofen administrations following the TAI treatment regimen improve pregnancy percentages in dairy cattle as a result of metabolic and endocrine milieu modulations.