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Endometrial epidermal growth factor (EGF) shows a cyclic change with two peaks on days 2-4 and days 13-14 of the estrous cycle. In repeat breeder cows, loss of the peaks has been associated with reduced fertility. By infusing seminal plasma (SP) and osteopontin (OPN) derived from SP and milk into the vagina, their EGF profile and fertility are restored. However, SP is difficult to obtain, and both SP and OPN can transmit infectious diseases. While OPN can be sourced from recombinant protein without this risk, recombinant bovine OPN (rOPN) expressed in Escherichia coli should be examined for its effects on the EGF profile, since it does not undergo posttranslational modification, which is important for its biological activity. In study 1, PBS, SP (0.5 mL), and rOPN (0.3 mg) were infused into the vagina at estrus (day 0) in 74, 37, and 105 repeat breeder Holstein cows, respectively, with an altered EGF profile. The endometrial EGF concentrations were measured on day 3. Some cows (n = 58, 20, and 83, respectively) were inseminated immediately before the infusion and then diagnosed for pregnancy between days 30 and 35. The normalization rate of the EGF profile and conception rate in the rOPN group (58.1 % and 47.0 %, respectively) were not significantly different from those in the SP group (62.2 % and 45.0 %, respectively) but higher than those in PBS group (29.7 % and 28.1 %, respectively) (P < 0.05). In study 2, repeat breeder cows with an altered EGF profile were infused with PBS (n = 18) and rOPN (n = 17), while fertile controls with a normal EGF profile (n = 18) were infused with PBS. Two or three embryos were transferred into cows on day 7 and then recovered on day 14. Embryo recovery rates of the rOPN and fertile groups were comparable (58.7 % vs. 58.3 %) but higher than that of the PBS group (58.7 % vs. 32.0 %) (P < 0.05). The embryo recovery rate of cows with normalized EGF profile was higher than that of cows with unnormalized EGF profile (64.4 % vs. 16.7 %) (P < 0.05). The embryo sizes of cows in the rOPN and fertile groups were comparable but larger than those in the PBS group (P < 0.05). However, the embryo size was not correlated to the corresponding endometrial EGF concentrations. In conclusion, rOPN without posttranslational modifications normalized the EGF profile in repeat breeder cows. Improved fertility by normalization of the EGF profile could be attributed partly to the increased embryo viability up to day 14., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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.

Endometrial epidermal growth factor (EGF) shows a cyclic change with two peaks on Days 2-4 and 13-14 during the estrous cycle. An altered (i.e., loss of the two peaks) profile has been linked to reduced fertility in repeat breeder cows. We previously demonstrated that a form of osteopontin (OPN), with a molecular weight of 29 kDa and found in bull seminal plasma (SP), normalized the EGF profile and restored fertility in repeat breeder cows. OPN has many molecular forms due to post-translational modifications and is abundant in bovine milk. The purpose of the present study was to investigate whether mOPN normalizes the endometrial EGF profile and restores fertility in repeat breeder dairy cows with an altered EGF profile. OPN was separated by one-step anion-exchange column chromatography from the whey of bovine milk. Purified mOPN was verified by Western blotting and peptide mass fingerprinting analyses. The OPN fraction showed three major protein bands of 61, 37 and 31 kDa (peptides I, II, and III, respectively) on SDS-PAGE. All three major bands were identified as OPNs by Western blotting and their tryptic peptide masses were matched at approximately 50, 40, and 10%, respectively, to the bovine OPN amino acid sequence by a peptide mass finger printing analysis. The three bands accounted for approximately 85% of the total protein content and 6-23 mg of OPN was obtained from 1 L of bovine milk. A lyophilized eluate containing 1.3 mg of mOPN (171 cows), 0.5 mL of frozen SP (62 cows), and PBS (84 cows) was infused at estrus into the vagina of repeat breeder cows with an altered EGF profile. Some of the cows treated with mOPN, SP, and PBS (46, 50, and 45 cows, respectively) were inseminated immediately before the infusion and then examined for pregnancy between Days 60 and 65. The rate at which mOPN to normalize the EGF profile (56.1%) was similar to that of SP (58.1%) and higher than that of PBS (23.8%) (P < 0.05). The conception rate after the infusion of mOPN (43.5%) was similar to that of SP (40.0%) and higher than that of PBS (22.2%) (P < 0.05). The present results indicate that the infusion of mOPN into the vagina is a treatment option for repeat breeder cows with an altered EGF profile. Further studies are needed to compare the capacity of the three OPN molecules in milk to normalize the EGF profile, together with their molecular characteristics due to post-translational modifications., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The endometrial epidermal growth factor (EGF) profile is an indicator of uterine function and fertility in cattle. The present study aimed to investigate the effects of heat stress on the endometrial EGF profile and fertility in lactating Holstein cows. The endometrial EGF profiles of 365 cows in the Hokkaido and Kyushu regions were examined between June and September (heat stress period, n = 211) and between October and January (control period, n = 154). EGF profiles were investigated using uterine endometrial tissues obtained by biopsy 3 days after estrus (Day 3). The proportion of cows with an altered EGF profile was higher between June and September than between October and January (41.2 vs. 16.2%, P < 0.05). The effects of rectal temperature on Days 0 and 3 on the endometrial EGF profile were also assessed in cows (n = 79) between June and September in the Kyushu region. A single embryo was transferred to cow on Day 7 to evaluate fertility (n = 67). Regardless of the rectal temperature on Day 3, the proportion of cows with an altered EGF profile was higher (64.1 vs. 30.0%, P < 0.05) and the pregnancy rate after embryo transfer (ET) was lower (26.7 vs. 51.4%, P < 0.05) in cows with a rectal temperature ≥ 39.5°C on Day 0 than in cows with a rectal temperature < 39.5°C on Day 0. The present results indicate that alterations in the endometrial EGF profile induced by an elevated body temperature on Day 0 contributed to reductions in fertility in lactating dairy cows during the heat stress period.

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.

Normal cows have 2 peaks in endometrial epidermal growth factor (EGF) concentrations on Days 2-4 and 13-14, and the absence of peaks has been linked to reduced fertility in repeat breeder (RB) cows. However, the timing of the peaks (i.e., day of examinations) was estimated for a few cows per cycle day. Therefore, the present study characterized EGF peaks and examined if the absence of peaks in RB cows indicate either peak loss or changed timing. In Study 1, 20 Holstein cows were examined for EGF concentrations between Days 1 and 6 using repeated biopsy of the uterine endometrial tissues. Sixteen cows exhibited increased EGF concentrations for 2-3 days between Days 2 and 5. All 16 cows exhibited increased EGF concentrations on Day 3. In Study 2, 10 cows were examined for EGF concentrations between Days 11 and 16. Increased EGF concentrations for 2-3 days were found in 7 cows between Days 12 and 15. All 7 cows exhibited increased EGF concentrations on Days 13 and 14. In Study 3, 12 RB cows were examined for endometrial EGF concentrations between Days 1 and 6. Four cows exhibited an increase of EGF concentrations on Days 3 and 4, whereas 8 cows (66.7%) exhibited low EGF concentrations throughout the study period. In conclusion, Days 3 and 13-14 are suitable days to examine a cyclic change of endometrial EGF concentrations. Further, low EGF concentrations on Day 3 in RB cows indicated an absence, but not altered timing, of the EGF peak.

A high incidence (about 70%) of alteration in endometrial epidermal growth factor (EGF) profile, i.e., loss of 2 peaks on days 2-4 and 13-14, has been linked to a reduced fertility in multiparous repeat breeder Holstein cows. However, the EGF profile in Holstein heifers and other breeds (types) of cattle has not been investigated. In study 1, EGF concentrations were determined using endometrial tissues obtained by biopsy on days 3, 7 and 14 from 84 fertile Holstein heifers to obtain a normal range and 53 repeat breeder Holstein heifers to estimate incidence of alterations in the EGF profile. In repeat breeder heifers, EGF concentrations were similar to fertile controls on 3 days and five animals (9.4%) had an altered EGF profile with EGF concentrations below the normal range on days 3 and 14. In study 2, EGF concentrations on day 3 were repeatedly examined from the nulliparous period to the third postpartum period in 28 Holstein (dairy) and 47 Japanese Black (beef) cattle. The effect of parity on EGF concentrations on day 3 was different between Holstein and Japanese Black cattle. In Japanese Black cows, the EGF concentrations were consistently high throughout the study period, while in Holstein cows, the EGF concentrations decreased after the second calving. In conclusion, unlike multiparous repeat breeder Holstein cows, an altered EGF profile may not be a major cause of repeat breeding in Holstein heifers, and the peak EGF concentrations around day 3 may decrease even in fertile populations of multiparous dairy cows, but not in beef cows.

Poor reproductive efficiency is a worldwide problem that has affected the dairy industry during the last several decades. In an attempt to explain the changes in reproductive physiology caused by high milk production, a model of elevated steroid metabolism in lactating dairy cows has been proposed. A slow increase in levels and low peak levels of estradiol (E₂) and progesterone (P₄) characterize endocrine changes in high producing cows. Similar changes have been reported in the repeat breeder cows. The abnormal changes in E₂ and P₄ concentrations of these cows may cause an improper uterine environment due to disturbed expression of growth factors and cytokines in the endometrium. This review focuses on the alteration in epidermal growth factor (EGF) profile in the endometrium during the estrous cycle. The normal cow has two peaks of EGF concentrations on days 2-4 and 13-14. Low concentrations of EGF on these days distinguished both high-producing and repeat breeder cows from normal cows. Alteration of the EGF profile could be found in 70 and 40% of the repeat breeder and high-producing cows, respectively. Treatment with a high dose of estradiol benzoate and an intravaginal progesterone-releasing device restored the normal EGF profile in about 70% of the affected cows. The cows having a normal EGF profile after treatment showed a higher pregnancy rate than the cows with the altered profile. Further studies to understand the etiology of the alteration in the EGF profile are needed to develop another treatment option and preventive management for this problem.

The objective of the present study was to examine the efficacy of a progestin-based treatment with a high dose of estradiol benzoate (EB) to normalize the epidermal growth factor (EGF) profile in the uterine endometrium and restore fertility in repeat breeder cows. Repeat breeder cows without peaks in their endometrial EGF concentrations on Days 3 and 14 of the estrous cycle were used throughout the study. The effect of 1 (standard dose), 2.5 and 5 mg of EB in a progestin-based treatment protocol (EB1, EB2.5 and EB5 treatments, respectively; n=5 for each group) on endometrial EGF concentrations was first examined. The EB1 and EB2.5 treatments in the repeat breeder cows produced a suppressed response in endometrial EGF compared with EB1 treatment in the fertile controls (n=5) and failed to restore the normal EGF profile during the next estrous cycle. However, EB5 treatment produced an increase in EGF concentrations similar to the fertile controls and normalized the endometrial EGF profile. The effects of the EB1 and EB5 treatments (n=30 for each group) on the endometrial EGF profile and fertility were then examined in the repeat breeder cows. The proportion of cows, with an EGF profile normalized by the treatments was higher in the EB5 group (66.7%) than in the EB1 (30.0%) and untreated control (13.3%; n=30) groups (P<0.01). The pregnancy rates of the cows having a normal EGF profile after treatment in the EB1 and EB5 groups were similar (88.9 and 85.0%, respectively) and higher than those of the cows having an abnormal profile within the same groups (19.0 and 30.0%, respectively, P<0.01). In summary, the endometrial response to EB in terms of the EGF concentration was suppressed in repeat breeder cows. A high dose (5 mg) of EB in a progestin-based treatment was found to be effective for restoration of a normal EGF profile and fertility in repeat breeder cows having lesser endometrial EGF concentrations on Days 3 and 14.

The present study characterized alterations of the endometrial epidermal growth factor (EGF) and examined potential relationships between normalization of the EGF and restoration of fertility in repeat breeder cows. In Experiment 1, endometrial tissues were obtained by biopsy on Days 3, 7 and 14 of the estrous cycle from 99 fertile dairy cows and normal ranges of uterine tissue EGF concentrations were determined. Then, eight fertile cows were examined for endometrial EGF concentrations on Days 3, 7 and 14 during the three consecutive estrous cycles. All eight cows had a normal EGF profile (EGF concentrations were within the normal ranges of all 3 days) in all three estrous cycles and endometrial EGF concentrations were similar among the three estrous cycles. In Experiment 2, 61 repeat breeder cows were examined EGF profile and 43 (70.5%) cows had altered EGF profiles compared with control profiles. A typical alteration was characterized by decreased concentrations on Days 3 and 14 and accounted for 86.0% of all alterations. When repeat breeder cows with altered EGF profiles (n=17) were left untreated, abnormality persisted in 14 (82.4%) cows at the second examination and only 3 animals became pregnant within the next two estrous cycles. Among 15 cows showing a typical alteration at the first examination, 11 (73.3%) cows had the same alteration until the third examination performed after two infertile inseminations following the second examination. When repeat breeder cows with altered EGF profiles (n=26) were given one of four therapeutic treatments, cows in which the EGF profile normalized after treatment had a greater pregnancy rate than those with altered profiles (11/14 animals versus 3/12 animals pregnant; P<0.05). In conclusion, suppressed endometrial EGF concentrations on Days 3 and 14 may be common alterations and persist between estrous cycles in repeat breeder cows. Potential relationship between normalization of the endometrial EGF profile and restoration of fertility was observed. Additional study to confirm results of the treatment is, however, necessary because the present study used a limited number of animals. Nevertheless, current data suggest that normalization of the EGF profile is one of the prerequisite factors to restore fertility.

The objective of the present study was to determine if abnormalities in the cyclic changes of endometrial EGF concentrations can be a diagnostic tool for repeat breeder cows. First, the profile of EGF concentrations during the estrous cycle was determined using endometrial tissues obtained from 31 Holstein cows after slaughter. Cyclic cows had two peaks of EGF concentrations. Then, endometrial tissues were obtained from 12 control and 20 repeat breeder cows by biopsy on Days 3, 7, and 14 of the same estrous cycle. Endometrial EGF concentrations in biopsied samples of the controls were similar to those found in slaughterhouse materials; they were high on Days 3 and 14 (9.2 and 10.4 ng/g tissue, respectively) and low on Day 7 (3.8 ng/g tissue). Concentrations of EGF in repeat breeder cows had a different profile; they were similar on Days 3, 7, and 14 (4.4, 3.4, and 4.0 ng/g tissue, respectively). In conclusion, changes in endometrial EGF concentrations were altered in repeat breeders; these alterations may be a potential diagnostic marker for repeat breeder cows.

Inadequate exposure to estradiol (E 2) and progesterone (P 4) may be the main causes of altered endometrial epidermal growth factor (EGF) profile, leading to reduced fertility in dairy cows. We hypothesized that PGF 2α administration at different timings of the estrous cycle and stages of follicular development could change the profile of steroid hormones between luteolysis and estrus. This results in reduction in the peak concentration of endometrial EGF on day 3 (day 0 = estrus) in the next estrous cycle. In study 1, lactating Holstein cows were treated with PGF 2α either on days 12–14 (selection phase group, n = 20) or on days 16–17 (control group, n = 24) of the estrous cycle. Blood samples were obtained before PGF 2α treatment, 24 and 48 h after treatment, and on the day of estrus and ovulation for E 2 and P 4 assays. Endometrial tissues were collected by biopsy on day 3 for EGF assays. The duration from PGF 2α treatment to both estrus and ovulation was longer in the selection phase group than in the control group (P < 0.05). The time between estrus and ovulation was longer in the selection phase group (P < 0.05). E 2 concentrations were higher in the control group on the day of estrus (P < 0.05). P 4 concentrations were not different between the groups. Endometrial EGF concentrations were lower in the selection phase group than in the control group (P < 0.05). In study 2, lactating Holstein cows were treated with PGF 2α either on day 12 (selection phase group, n = 76) or day 16 (control group, n = 80). They were subjected to artificial insemination (AI) at estrus, and those that failed to ovulate by 24h after the first AI were subjected to second AI. On day 3, endometrial EGF concentration was determined. At estrus and on days 3 and 7, blood was collected for E 2 and P 4 assay from 20 randomly selected cows in each group before PGF 2α treatment. The cows in the selection phase group exhibited lower EGF concentration, proportion of cows with normal EGF profile, and conception rate than cows in the control group (P < 0.05). On the day of estrus, E 2 concentrations tended to be lower in the selection phase group than in the control group (P = 0.08). P 4 concentrations were not different between the groups. These findings suggest that low E 2 concentrations at estrus and low P 4 concentrations for a prolonged period due to premature termination of CL in the absence of a dominant follicle are potential causes of altered endometrial EGF profile in dairy cows. • Treatment using PGF2α during follicle selection of the second follicle wave. • Extended the time from treatment to estrus and ovulation. • Reduced the proportion of cows ovulating within 24 h after AI. • Reduced the endometrial EGF concentrations on the next cycle and conception rate. [ABSTRACT FROM AUTHOR]