Identification of bovine seminal plasma proteins with an activity to normalize endometrial epidermal growth factor concentrations in repeat breeder cows

Epidermal growth factor (EGF) concentrations in the bovine uterus show two peaks on Days 2-4 and 13-14 during the estrous cycle in normal cows; however, these peaks were not found in about 70% of Holstein repeat breeder cows. We have demonstrated the effect of seminal plasma (SP) to normalize the endometrial EGF concentrations on Day 3 and restore fertility in repeat breeder cows. The objective of this study was to identify SP protein(s) with the activity of normalizing the endometrial EGF concentrations. Semen was collected from 5 Holstein bulls and pooled SP obtained from 30 ejaculates were used for this study. The SP protein were separated by gel filtration and 2-dimensional electrophoresis. SP fractions with molecular weight of 16-29 kDa and pI5.8-7.0 showed an activity to normalize the endometrial EGF concentrations on Day 3. Then, protein spots in these area on electrophoresis gels were extracted and subjected to liquid chromatography with tandem mass spectrometry analysis. Twelve protein spots that include four spots of osteopontin (OPN) with different molecular weights and isoelectric points were identified. Protein extracts of one of these OPN spots normalized the endometrial EGF concentrations on Day 3 in 41.9% of repeat breeder cows. The present results indicated that OPN may be the molecule responsible for the activity normalizing the EGF concentrations on Day 3 in the endometrium of repeat breeder cows

Effect of seminal plasma infusion into the vagina on the normalization of endometrial epidermal growth factor concentrations and fertility in repeat breeder dairy cows

Epidermal growth factor (EGF) concentrations in the uterus show two peaks on days 2-4 and 13-14 during the estrous cycle in fertile cows. Loss of the two peaks has been linked to reduced fertility in repeat breeder cows. This study aimed to examine the effect of seminal plasma (SP) on normalizing endometrial EGF concentrations and restoring fertility in repeat breeder cows with low EGF concentrations on day 3. In study 1, we examined the effect of the deposition sites (the vagina and uterus) of SP on the endometrial EGF concentrations in repeat breeder cows. SP infusion into the vagina, but not uterus, on the first day of the estrus cycle (day 0) normalized the endometrial EGF concentrations (>= 4.7 ng/g tissue weight) on day 3. In study 2, the effect of SP volume (0.5 and 10 ml of SP and 0.5 ml of SP diluted to 10 ml) on EGF concentrations was examined. All groups with SP infusion had increased EGF concentrations on day 3, and cows with 10 ml of SP and 0.5 ml of SP diluted to 10 ml showed the highest levels of EGF concentrations. In study 3, we examined the effect of SP infusion on fertility. SP infusion normalized two peaks of endometrial EGF concentrations in about 60% of repeat breeder cows and produced more pregnancies than the controls (44.4 vs. 19.4%). Therefore, we concluded that SP may contain an activity to normalize the EGF profile and restore fertility in repeat breeder cows with altered EGF profiles

Leptin receptor expression and its change in association with the normalization of EGF profile after seminal plasma treatment in repeat breeder dairy cows

Factors associated with high milk production levels have been linked to alterations in the endometrial epidermal growth factor (EGF) profile, a cause of reduced fertility in dairy cows. Therefore, we examined the leptin system that connects nutritional status and reproduction in dairy cattle related to reduced fertility in repeat breeder cows. Plasma leptin concentrations were measured in 18 heifers, 20 high-yielding control cows, and 26 repeat breeder cows, showing an altered EGF profile. Then, all repeat breeder cows were infused with seminal plasma (SP) into the vagina at the next estrus to normalize the EGF profile, while heifers and control cows were infused with vehicle alone. All animals were examined for EGF profiles. Eighteen repeat breeder cows, nine heifers, and nine control cows were also determined for leptin receptor (Ob-R) expression levels in the estrous cycle before and after the infusion. SP normalized the EGF profile in 53.8% of the repeat breeder cows. Leptin concentrations were similar in all groups, regardless of the treatment results for the EGF profile. In contrast, Ob-R levels in repeat breeder and control cows were similar and higher than those in heifers before SP treatment. Ob-R in repeat breeders showing a normal EGF profile after treatment decreased to an intermediate level between heifers and control cows and may provide a clue to take measures against repeat breeding in dairy cows