Fertility In Repeat Breeder Cows Research Articles

Identification of an osteopontin structural element for the restoration of a normal endometrial epidermal growth factor (EGF) profile determined by the EGF concentration on day 3 of estrous cycle and pregnancy outcome in repeat breeder dairy cows

The loss of a cyclic change with two peaks of increased endometrial epidermal growth factor (EGF) concentration on days 2−4 and 13−14 during the estrous cycle has been linked to low fertility in repeat breeder (RB) cows. We have shown that an intravaginal infusion of osteopontin (OPN) restored the EGF profile in RB cows. The present study aimed to determine a structural element of OPN to restore the normal EGF profile and fertility. Holstein RB cows were diagnosed the EGF profile by a single examination of the endometrial EGF concentration on day 3 of the estrous cycle. Those with an altered EGF profile were intravaginally infused with OPN and its fragments on the day of insemination (day 0); the concentration of endometrial EGF was measured on day 3, and pregnancy was diagnosed on days 30−35. In Study 1, recombinant OPN (rOPN) (16 nmol), thrombin-cleaved N- and C-terminal fragments of rOPN (N-rOPN and C-rOPN, respectively), and a combination of these fragments (Th-rOPN) were infused (n = 13−20). The restoration rate of the normal EGF profile of the N-rOPN group (25.0%) was a level in between the C-rOPN group (7.7%) and both the rOPN (55.6%) and Th-rOPN (64.3%) groups. In Study 2, PBS (n = 47), rOPN (9.5 nmol, n = 83), and peptides of integrin binding motifs, GRGDSVAYGLK (peptide 1; 32, 320, and 1,600 nmol), GRGDS (peptide 2; 320 and 1,600 nmol), and SVAYGLK (peptide 3; 320 and 1,600 nmol), were infused (n = 20−25). Restoration rates of the normal EGF profile of peptide 1 (320 and 1,600 nmol) and peptide 3 (1,600 nmol) groups (44.0−56.3%) were comparable with those of the rOPN group (63.9%) and higher than those of the PBS group (15.6%). Restoration rates of the other groups were similar to those of the PBS group. Additional cows received infusions to determine the effect on fertility. Conception rates of the peptide 1 (320 and 1,600 nmol; n = 50 each), peptide 3 (1,600 nmol; n = 55), and rOPN (n = 111) groups (41.8−50.0%) were comparable and higher than that of the PBS group (21.6%, n = 75). In Study 3, PBS (n = 24), peptide 1 (320 nmol; n = 78), and GRGESVAYGLK peptide (peptide 4; 320 and 1,600 nmol; n = 50 and 26, respectively) were infused. Restoration rates of the normal EGF profile of peptide 4 and PBS groups (16.0−19.2%) were comparable and lower than those of the peptide 1 group (44.9%). Thus, the SVAYGLK motif may be an OPN structural element to restore the normal EGF profile and fertility in RB cows, and the RGD motif may enhance its effect.

Effects of milk osteopontin on the endometrial epidermal growth factor profile and restoration of fertility in repeat breeder 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.

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.

The ovulatory response to human chorionic gonadotropin administration on day 4 post timed artificial insemination improved fertility in repeat breeder cows

This study focused on determining the success of inducing endogenous progesterone source (by formation of accessory corpus luteum) by human chorionic gonadotropin (hCG) administration on day 4 post timed artificial insemination (TAI) and on evaluating the effect of the day of hCG administration (day 4 or 5 post TAI) on pregnancy rates in repeat breeder cows according to their responses to hCG administrations. Non-pregnant cyclic lactating Holstein dairy cows with more than three services despite no clinical reproductive abnormalities were enrolled in the study. All cows (n = 192) received the Ovsynch protocol (GnRH-7d-PGF2α-56h-GnRH-16/18h-TAI) and hCG administration (1500 IU, i.m.) was performed in group hCG4 (n = 64) on day 4 and in group hCG5 (n = 68) on day 5 post TAI. The control group (CON, n = 60) did not receive any hCG treatment. The responses (ovulation rate) to hCG (~64%) were similar on days 4 and 5 post TAI. Pregnancy per AI (P/AI) at 31 days was similar among the groups (48.3% in CON, 48.4% in hCG4, and 42.6% in hCG5). Regardless of the day of the treatment (hCG4 + hCG5), the cows had an ovulatory response to hCG (54.1%), and a greater P/AI (P = 0.007) at 31 days than those without a response (29.8%). The administration of hCG on either day 4 or 5 post TAI did not increase overall pregnancy rates, however, the ovulatory response to the post TAI hCG administration on day 4 improved fertility in repeat breeder cows. Further studies are needed with a larger number of animals to reveal this potentiallly beneficial effect.

Treatment with an elevated dose of the GnRH analogue dephereline in the early luteal phase improves pregnancy rates in repeat-breeder dairy cows

This study sought to establish whether treatment with the GnRH analog dephereline in the early luteal phase could be useful to improve fertility in repeat-breeder cows (>3 inseminations). Dephereline was given 5–7 days after insemination, and effects of both a single and elevated (2.5 times) dephereline dose on pregnancy and subsequent embryo survival determined in repeat-breeder lactating dairy cows. The study population comprised 810 cows: 399 repeat-breeder cows and 411 non repeat breeders. To determine the effects of treatment on the pregnancy rate, three study groups were established: Control, no treatment (n = 269), DEPH, single dephereline dose (100 μg) (n = 270), or 2.5DEPH, 2.5 dephereline doses (250 μg) (n = 271). Recorded pregnancy rates in these groups were 28.6% (77/269), 31.9% (86/270) and 39.1% (106/271), respectively. Binary logistic regression analysis revealed a significant effect of the interaction treatment x repeat breeder on the pregnancy rate. This meant that the single dose and control treatments led to a pregnancy likelihood of repeat-breeder cows reduced by factors of 0.47 and 0.34, respectively, when compared to the 2.5 dose treatment as reference. Treatment had no effects on non repeat breeders. Based on odds ratios, treatment given either as a single or 2.5 dose increased the probability of an additional corpus luteum (more corpora lutea than embryos) in pregnant cows by factors of 5 or 5.4, respectively. Treatment had no effect on embryo survival. Our findings support the hypothesis that dephereline treatment in the early luteal phase after AI improves fertility in repeat-breeder cows. A dephereline dose 2.5 times higher than normal led to improved pregnancy rates, thus overcoming the impacts of repeat-breeder syndrome. Importantly, both the single and 2.5 dephereline doses increased the chances of pregnant cows having an additional corpus luteum, likely favoring embryo survival.

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.

Short communication: Repeat breeder cows with fluid in the uterine lumen had poorer fertility

The objective of the present study was to test the association between the presence of fluid in uterine lumen and the fertility of repeat breeder cows (RBC). Grazing Holstein cows that were clinically normal and were not pregnant after at least 3 consecutive inseminations were defined as RBC and included in the study (n = 358). A cut-point ≥2 mm of uterine lumen visible by ultrasonography was used as indicative of the presence of fluid in the uterus. The effect of this fluid on the fertility of RBC was assessed by mixed logistic regression. Twenty percent of the RBC had ≥2 mm of fluid in their uterus, and this finding was associated with a marked reduction in the odds of conception (odds ratio = 0.46). Additional intrauterine sampling revealed that 24.0% of RBC showed signs of subclinical endometritis [≥5% neutrophils in endometrial cytology (SCE)]. However, we found no agreement between the finding of uterine fluid and the diagnosis of SCE (kappa coefficient = 0.03, 95% confidence interval = -0.07 to 0.14). In conclusion, the presence of fluid (≥2 mm) in uterine lumen, detected by ultrasonography, was associated with lower fertility in RBC, but was not related to the diagnosis of SCE. Thus, ultrasonographic evaluation is an important tool to assist in assessment of RBC.

Comparison of serum leptin, glucose, total cholesterol and total protein levels in fertile and repeat breeder cows

In the present study we measured serum glucose, leptin, total cholesterol and total protein concentrations in repeat breeder cows and compared them with fertile cows. For this aim, 20 repeat breeder cows and 20 fertile cows were used as material. Repeat breeder cows were found to have lower levels of leptin and glucose as compared with fertile ones. No significant differences in total cholesterol and total protein levels were observed between the two groups. No significant correlation of leptin with glucose, total cholesterol and total protein was observed in fertile and repeat breeder cows. Low concentrations of glucose and leptin can have some effects on reproductive problems as repeat breeder and help to understand potential mechanisms impairing fertility in repeat breeder cows.

A Three-day PGF&lt;sub&gt;2α&lt;/sub&gt; Plus eCG-based Fixed-time AI Protocol Improves Fertility Compared with Spontaneous Estrus in Dairy Cows with Silent Ovulation

This study was designed to test the treatment with prostaglandin F2α (PGF2α) plus equine chorionic gonadotrophin (eCG) and GnRH 48 h later (PEG protocol) followed by fixed-time AI (FTAI) in dairy cows with silent ovulation (cows with a mature corpus luteum and no signs of estrus detected in the preceding 21 days). In Experiment I, ovulation following the PEG protocol monitored in 24 cows with silent ovulation was recorded in 83% of the cows 36 h after GnRH treatment. In Experiment II, control cows were artificially inseminated during spontaneous estrus (4615 AI), while cows in the PEG group (1266 AI) were subjected to FTAI 24 h after GnRH treatment. Binary logistic regression revealed a significant effect of the interactions of treatment by season, by parity or by repeat breeding syndrome (>3 AI) on the conception rate. The conception rate was negatively affected by the warm season and age in controls but not in treated cows, whereas repeat breeder cows in the control and PEG groups were less (by a factor of 0.7) or more (by a factor of 1.5) likely to become pregnant, respectively, than the remaining animals. Moreover, the likelihood of twin pregnancy was lower in multiparous PEG (by a factor of 0.4) cows than in the remaining cows. This protocol, besides overcoming the negative effects of heat stress and age on the conception rate, increased fertility in repeat breeder cows compared with spontaneous estrus. Moreover, this treatment regimen reduced the twin pregnancy rate in multiparous cows.

A Progestin-Based Treatment with a High Dose of Estradiol Benzoate Normalizes Cyclic Changes in Endometrial EGF Concentrations and Restores Fertility in Repeat Breeder Cows

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.

Potential relationship between normalization of endometrial epidermal growth factor profile and restoration of fertility in repeat breeder cows

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.

リピートブリーダー牛の卵管通気検査とその後の受胎成績

リピートブリーダー牛の卵管通気検査とその後の受胎成績