Evidence that interferon-tau secreted from Day-7 embryo in vivo generates anti-inflammatory immune response in the bovine uterus.

Recent studies suggest that Day-7 bovine embryo starts to communicate with the uterine epithelium through interferon-tau (IFNT) signaling. However, immune modulatory role of IFNT in the uterus just after the embryo moves from the oviduct is unclear. We aimed to examine the hypothesis that Day-7 bovine embryo secretes IFNT in the uterus, which induces anti-inflammatory response in immune cells. The uterine flush (UF) with multiple embryos was collected from Day-7 donor pregnant cows and peripheral blood mononuclear cells (PBMCs) were then cultured in UF. Transcripts detected in PBMCs revealed that UF from pregnant cows down-regulated pro-inflammatory cytokines (TNFA, IL1B) and up-regulated anti-inflammatory cytokine (IL10) expression, with activation of interferon-stimulated genes (ISGs; ISG15, OAS1) as compared with UF from non-pregnant cows. An addition of specific anti-IFNT antibody to the UF inhibited the effect on PBMCs, indicating that IFNT is a major factor for such immune modulation. The observation that conditioned media from bovine uterine epithelial cells both stimulated with IFNT in vitro and supplemented with fresh IFNT induced similar PBMCs gene expression, confirming that IFNT directly acts on this immune crosstalk. This study shows that IFNT secreted from Day-7 embryo in vivo generates anti-inflammatory response in immune cells, which may provide immunological tolerance to accept the embryo.

Long-term changes in plasma anti-Müllerian hormone concentration and the relationship with superovulatory response in Japanese Black cattle.

The concentration of circulating anti-Müllerian hormone (AMH) in cattle is a useful endocrine marker for ovarian response to superovulation. Although the AMH concentration undergoes little variation throughout the estrous cycle, its long-term changes remain incompletely understood. Here, we investigated the relationship between superovulation response and plasma AMH concentration in Japanese Black cattle and the long-term changes in plasma AMH concentration of embryo donor cows and heifers. The median, 25th percentile, and 75th percentile of AMH concentrations in 222 mature animals were 0.265, 0.118, and 0.488 ng/ml, respectively. The numbers of ova/embryos, fertilized embryos, and transferable embryos in a total of 295 superovulations were significantly different among the H (AMH ≥ 0.488 ng/ml), M (AMH 0.487-0.119 ng/ml), and L (AMH ≤ 0.118 ng/ml) groups. AMH concentrations during repeated superovulation in ten donor cows were significantly decreased after the third treatment. In heifers, the highest AMH concentration was observed in individuals during 2-13 months of age, with considerable individual variability. AMH concentrations of heifers at 10 or 11 months correlated with the number of ova/embryos during superovulation at 13-18 months (r = 0.641, P < 0.05). These results suggest that the 25th and 75th percentile values of AMH concentration would give a useful rough estimate of ovarian response; however, repeated superovulation may reduce the predictive accuracy of single measurements of AMH concentration. It would be possible to evaluate AMH concentration in heifers after approximately 11 months of age.

Day-7 embryos generate an anti-inflammatory immune response in peripheral blood immune cells in superovulated cows.

PROBLEM: Recently, we demonstrated both in vitro and in vivo that an immunological crosstalk between Day-7 embryo and immune cells exists locally in the uterus in cows. The peripheral immune response to early embryos at Day-7 of pregnancy in cows remains largely unknown. Thus, we aimed to investigate the response of peripheral blood immune cells in the presence of multiple Day-7 embryos in the uterus in donor cows of embryo transfer program. METHOD OF STUDY: Superovulated cows were either inseminated (n = 13) at 12-18 hours post-estrus or remained without inseminations (n = 6). Blood was collected following insemination (Day-1) and immediately after uterine flushing (Day-7). Polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood for analysis of gene expression. RESULTS: Interferon-stimulated genes (ISGs: ISG15 and OAS1) were increased in both PMNs and PBMCs, with up-regulation of PTGES and anti-inflammatory cytokines (TGFB1 and IL10) expressions at Day-7 of post-inseminations in cows, when compared to those at Day-7 in non-inseminated cows. CONCLUSION: The findings indicate that the presence of multiple embryos in the uterus generates an anti-inflammatory immune response in peripheral blood immune cells at Day-7 of pregnancy in cows, which might help to accept semi-allogenic embryo in the uterus.

Effects of genetic background on responses to superovulation in Japanese Black cattle.

We investigated the effects of genetic background on the responses to superovulation in Japanese Black cattle. The genotype frequencies of GRIA1 and FSHR relating to ovulation and follicular development in each of the major bloodlines-Tajiri, Fujiyoshi, and Kedaka-were analyzed. The Tajiri line had the lowest frequency of G allele homozygosity of c.710A>G in GRIA1 among the three bloodlines, and deviation from Hardy-Weinberg equilibrium was detected. Genotype frequencies of c.337C>G, c.871A>G, and c.1973C>G in FSHR were in Hardy-Weinberg equilibrium in all bloodlines. The results of generalized linear mixed-model analyses showed that farm, levels of plasma anti-Müllerian hormone (AMH) concentration, age in months, repeated superovulation, c.337C>G in FSHR, and bloodlines had significant effects on the responses to superovulation. The number of transferable embryos in the group heterozygous for c.337C>G in FSHR was significantly higher than that in the group homozygous for the C allele. The Kedaka line showed a significantly higher number of ova/embryos, fertilized embryos, and transferable embryos than the Tajiri and Fujiyoshi lines. The concentration of circulating AMH is a useful endocrine marker for antral follicle counts. This study revealed the effects of genetic background on the responses to superovulation using levels of plasma AMH concentration as a covariate. The prominent effect of genetic background on superovulation in the Kedaka line requires additional studies to confirm the genomic regions and polymorphisms that are involved in the trait.

Active immunization against inhibin improves superovulatory response to exogenous FSH in cattle.

The effect of active immunization against inhibin on the response to superovulatory treatment by porcine FSH (pFSH) was investigated in cattle. Japanese black cows were sc injected with 1 mg of porcine inhibin alpha-subunit fragment (1-26) conjugated with rabbit serum albumin (inhibin-immunized group; n=14) or rabbit serum albumin alone (control group; n=12) in Freund's complete adjuvant. Booster injections (half the amount of the primary injection) were given 35 and 70 days after the primary injection. All cows were superovulated three times with pFSH. Three days after each injection of the antigen, a progesterone-releasing intravaginal device (CIDR-B) was inserted vaginally into all animals and left in place for 10 days. Forty-eight hours before CIDR-B removal, all animals were sc injected with 30 mg pFSH dissolved in 40% polyvinylpyrrolidone, and im injected with 750 microg of PGF2alpha at CIDR-B removal. Cows were artificially inseminated twice during estrus, and ova or embryos were collected 7 or 8 days after estrus. The number of corpora lutea, the number of ova or embryos and the number of transferable embryos in inhibin-immunized cows (12.1+/-1.2, 11.1+/-1.3 and 6.2+/-1.0, respectively) were significantly greater than those in the controls (8.2+/-1.0, 5.7+/-1.1 and 3.1+/-0.7, respectively). These results indicate that active immunization against inhibin enhanced ovarian response to the usual superovulatory treatment in cattle. Therefore, immunization against inhibin may be a useful approach for improving the response to superovulation in cattle.