Postovulatory treatment with GnRH on day 5 reduces pregnancy loss in recipients receiving an in vitro produced expanded blastocyst.

The present study tested the hypothesis that administration of GnRH on day 5 of the estrous cycle in embryo transfer (ET) recipients would increase progesterone (P4) concentrations, embryo size, and improve fertility. Holstein and cross-bred Holstein heifers (n = 1562) were synchronized using a modified 5-day CIDR-Synch protocol as follows (All AM treatments): D-8, CIDR inserted; D-3, CIDR removed and PGF2α (500 µg cloprostenol) treatment; D-2, second PGF2α; D0, GnRH (G1, 100 µg gonadorelin acetate) to induce ovulation. On D5 in the afternoon, heifers were assigned in a completely randomized design to one of two treatments: Control (untreated) or GnRH (200 µg). Transfer of day 7 fresh IVP embryos was performed between D6 and D8 after G1. Data collected from each heifer included: embryo stage and quality, body condition score, technician performing ET, interval from G1 to ET, and number of previous transfers. All heifers were evaluated by transrectal ultrasonography on D5, D33, and D60 and a subset of heifers was scanned on D12 (n = 718; to determine ovulation to treatment) and another subset on D33 (n = 295; 16 s video to determine embryo and amniotic vesicle size). Serum P4 was determined from a subset of heifers on D12 (n = 467) and on D21 (n = 837) and pregnancy specific protein B (PSPB) on D28 (n = 843). Pregnancies per ET (P/ET) were analyzed by logistic regression and continuous outcomes by ANOVA. Ovulation to D5 GnRH, defined by the presence of an accessory CL on D12, was 83.9% (302/360) in GnRH-treated heifers vs. 3.3% (12/358) in Controls (P < 0.001). On D12, P4 was greater (P < 0.001) in GnRH-treated heifers (7.2 ±â€¯0.1 ng/ml) vs Controls (6.0 ±â€¯0.1 ng/ml). There was greater P/ET at D33 and D60 of pregnancy for Stage 7 than Stage 6 embryos. Treatment with GnRH did not alter P/ET with either embryo stage but decreased pregnancy loss between D33 and D60 in heifers receiving Stage 7 embryos. Presence of an accessory CL at the D33 pregnancy diagnosis was associated with a larger reduction in pregnancy loss from D33 to D60 in recipients of Stage 7 embryos (11.6 vs 27.6%). Although there was no GnRH effect on embryo size, the presence of an accessory CL was associated (P < 0.05) with larger amniotic vesicle volume in recipients of Stage 7 embryos. In addition, greater PSPB was linked to greater amniotic vesicle volume (P = 0.01) and to reduced pregnancy loss (P < 0.0001). In conclusion, treatment with GnRH on D5 caused ovulation and formation of an accessory CL, increased circulating P4, and reduced pregnancy loss in heifers receiving a Stage 7 but not a Stage 6 IVP embryo.

Mechanisms for rescue of corpus luteum during pregnancy: gene expression in bovine corpus luteum following intrauterine pulses of prostaglandins E1 and F2α.

In ruminants, uterine pulses of prostaglandin (PG) F2α characterize luteolysis, while increased PGE2/PGE1 distinguish early pregnancy. This study evaluated intrauterine (IU) infusions of PGF2α and PGE1 pulses on corpus luteum (CL) function and gene expression. Cows on day 10 of estrous cycle received 4 IU infusions (every 6 h; n = 5/treatment) of saline, PGE1 (2 mg PGE1), PGF2α (0.25 mg PGF2α), or PGE1 + PGF2α. A luteal biopsy was collected at 30 min after third infusion for determination of gene expression by RNA-Seq. As expected, IU pulses of PGF2α decreased (P < 0.01) P4 luteal volume. However, there were no differences in circulating P4 or luteal volume between saline, PGE1, and PGE1 + PGF2α, indicating inhibition of PGF2α-induced luteolysis by IU pulses of PGE1. After third pulse of PGF2α, luteal expression of 955 genes were altered (false discovery rate [FDR] < 0.01), representing both typical and novel luteolytic transcriptomic changes. Surprisingly, after third pulse of PGE1 or PGE1 + PGF2α, there were no significant changes in luteal gene expression (FDR > 0.10) compared to saline cows. Increased circulating concentrations of the metabolite of PGF2α (PGFM; after PGF2α and PGE1 + PGF2α) and the metabolite PGE (PGEM; after PGE1 and PGE1 + PGF2α) demonstrated that PGF2α and PGE1 are entering bloodstream after IU infusions. Thus, IU pulses of PGF2α and PGE1 allow determination of changes in luteal gene expression that could be relevant to understanding luteolysis and pregnancy. Unexpectedly, by third pulse of PGE1, there is complete blockade of either PGF2α transport to the CL or PGF2α action by PGE1 resulting in complete inhibition of transcriptomic changes following IU PGF2α pulses.

Physiological mechanisms involved in maintaining the corpus luteum during the first two months of pregnancy.

Maintenance of the corpus luteum (CL) during pregnancy is essential for continuing the elevated circulating progesterone (P4) that is required to maintain pregnancy. The mechanisms that protect the CL during early pregnancy when the non-pregnant animal would typically undergo CL regression have been extensively investigated. It is clear uterine prostaglandin F2α (PGF) causes regression of the CL in non-pregnant ruminants and that maintenance of the CL during early pregnancy is dependent upon secretion of interferon-tau (IFNT) from the elongating embryo. A number of specific mechanisms appear to be activated by IFNT. Most studies indicate that there is an inhibition of oxytocin-induced secretion of uterine PGF. There is also evidence for increased resistance to PGF action, perhaps due to secretion of PGE2 and PGE1 or direct endocrine actions of circulating IFNT. These mechanisms occur concurrently and each may help to maintain the CL during the first month of pregnancy. However, during the second month of pregnancy, IFNT is no longer secreted by the embryo. Attachment of the embryo to the uterus and subsequent placentome development have been linked to silencing of expression from the IFNT gene. In addition, there is some evidence that oxytocin responsiveness of the uterus returns during the second month of pregnancy leading to substantial basal secretion of PGF and perhaps PGF pulses. There is also no evidence that the CL during the second month of pregnancy is resistant to the actions of PGF as observed during the first month. Thus, this manuscript attempts to compare the mechanisms that maintain the CL during the first and second months of pregnancy in ruminants and provides a new, speculative, physiological model for maintenance of the CL during month two of pregnancy that is distinct from the previously-described mechanisms that maintain the CL during the first month of pregnancy.

Effect of feeding rumen-protected methionine on productive and reproductive performance of dairy cows.

The objectives of this study were to evaluate the effects of daily top-dressing (individually feeding on the top of the total mixed ration) with rumen-protected methionine (RPM) from 30 ± 3 until 126 ± 3 Days in milk on productive and reproductive performance in lactating dairy cows. A total of 309 lactating dairy Holstein cows (138 primiparous and 171 multiparous) were randomly assigned to treatment diets containing either RPM (21.2 g of RPM + 38.8 g of dried distillers grain; 2.34% Methionine [Met] of metabolizable protein [MP]) or Control (CON; 60 g of dried distillers grain; 1.87% Met of MP). Plasma amino acids were evaluated at the time of artificial insemination (AI) and near pregnancy diagnosis. Milk production and milk composition were evaluated monthly. Pregnancy was diagnosed on Day 28 (by Pregnancy-specific protein B [PSPB]), 32, 47, and 61 (by ultrasound) and sizes of embryonic and amniotic vesicle were determined by ultrasound on Day 33 after AI. Feeding RPM increased plasma Met at 6, 9, 12, and 18 hours after top-dressing with a peak at 12 hours (52.4 vs 26.0 µM; P < 0.001) and returned to basal by 24 hours. Cows fed RPM had a small increase in milk protein percentage (3.08 vs 3.00%; P = 0.04) with no differences on milk yield and milk protein yield. Additionally, in multiparous cows, RPM feeding increased milk protein (3.03 vs 2.95%; P = 0.05) and fat (3.45 vs 3.14%; P = 0.01) percentages, although no effects were observed in primiparous cows. In multiparous cows fed RPM, pregnancy loss was lower between Days 28 to 61 (19.6 [10/51] vs. 6.1% [3/49]; P = 0.03) or between Days 32 to 61 (8.9 [4/45] vs. 0 [0/0] %; P = 0.03), although, there was no effect of treatment on pregnancy loss in primiparous cows. Consistent with data on pregnancy loss, RPM feeding increased embryonic abdominal diameter (P = 0.01) and volume (P = 0.009) and amniotic vesicle volume (P = 0.04) on Day 33 of pregnancy in multiparous cows but had no effect on embryonic size in primiparous cows. Thus, the increase in plasma Met concentrations after feeding RPM was sufficient to produce a small increase in milk protein percentage and to improve embryonic size and pregnancy maintenance in multiparous cows. Further studies are needed to confirm these responses and understand the biological mechanisms that underlie these responses as well as the timing and concentrations of circulating Met that are needed to produce this effect.

Proposal of a new model for CL regression or maintenance during pregnancy on the basis of timing of regression of contralateral, accessory CL in pregnant cows.

In bovine pregnancy, regression or maintenance of the corpus luteum (CL) is mediated through local communication pathways between embryo, uterus, and ovary with Days 16 to 25 of pregnancy generally recognized as the pivotal period determining either luteolysis or prevention of luteolysis. To evaluate this concept, accessory CL was generated by treating Holstein lactating dairy cows (n = 718) with GnRH on Day 5 of the first follicular wave to produce an accessory CL on the ovary either contralateral or ipsilateral to the gravid horn. In pregnant cows, 66.2% (86/130) of contralateral CL regressed by Day 75 of pregnancy, whereas few ipsilateral accessory CL regressed (11.9%; 8/67), on the basis of similar criteria (P < 0.0001). As hypothesized, some contralateral CL regressions (22/86 = 25.6%) happened on Days 19 to 25 of pregnancy. However, most contralateral CL regressions (64/86 = 74.4%) happened later than expected, from Days 33 to 60 of pregnancy. Later contralateral CL regression was more common in primiparous (84.3%) than multiparous (60.0%; P = 0.02) cows. Early accessory contralateral CL regression (Days 19-25) may be related to lack of exposure of the contralateral horn to interferon tau from the elongating embryo because pregnant cows without early accessory CL regression had a smaller uterine volume than nonpregnant cows or pregnant cows that had early accessory CL regression (128.4 ± 3.9 vs. 147.0 ± 3.8 vs. 143.6 ± 10.9 mm3, respectively; P = 0.003). These results indicate that there is a second distinct period for CL protection during bovine pregnancy from Days 30 to 60 and implicate local and not systemic pathways in occurrence or prevention of luteolysis during both the early (≤25 days) and later (≥33 days) critical periods since accessory contralateral CL regressed whereas the accessory ipsilateral CL of pregnancy remained.

Pivotal periods for pregnancy loss during the first trimester of gestation in lactating dairy cows.

Loss of pregnancy can occur at many different stages of gestation and for a variety of causes but clearly produces a negative impact for reproductive and economic performances of dairy herds. This review describes four pivotal periods for pregnancy loss during the first trimester of gestation and discusses possible causes for pregnancy failure during these periods. The first period occurs during the first week after breeding with lack of fertilization and death of the early embryo producing major losses in pregnancy, particularly under specific environmental and hormonal conditions. In general, 20%-50% of high-producing lactating dairy cows have already experienced pregnancy loss during the first week of gestation with methods to decrease pregnancy loss during this period targeting improved oocyte quality by alleviating heat stress, inflammatory diseases, and body condition loss, and by increasing progesterone concentrations during preovulatory follicle development. The second pivotal period, from Days 8 to 27, encompasses embryo elongation and the classical "maternal recognition of pregnancy" period with losses averaging ∼30% but with surprising variation between farms (25%-41%). Maintenance of the CL of pregnancy is produced by the embryonic signal interferon-tau and alteration in uterine secretory patterns of prostaglandins F2α, E1, and E2. Failures or delays in trophoblast elongation and/or embryonic development result in loss of pregnancy during the second pivotal period possibly due to suboptimal histotroph. The third pivotal period is during the second month of pregnancy, Days 28 to 60, with losses of ∼12% based on a summary of published results from more than 20,000 pregnancies in high-producing dairy cows. Delays or defects in development of the chorioallantoic placentomes or embryo result in CL regression or embryo death during this pivotal period. Finally, a fourth period during the third month of pregnancy has reduced pregnancy losses (∼2%), compared with the first three periods but can be elevated in some cows, particularly in those carrying twins in the same uterine horn. Thus, there are varied causes for pregnancy losses during each pivotal period that correspond to key physiological changes in the embryo, uterine environment, and ovary. Similarly, strategies to reduce these losses are likely to require a multifaceted approach using rational methods that target the critical physiology in each pivotal period.

Effect of uterine size on fertility of lactating dairy cows.

There are multiple reasons for reduced fertility in lactating dairy cows. We hypothesized that one cause of reduced fertility could be the overall size of the reproductive tract, particularly the uterus, given well-established uterine functions in many aspects of the reproductive process. Thus, the objectives of this study were to evaluate the variability in uterine size in primiparous and multiparous dairy cows and to analyze whether there was an association between uterine size and fertility, particularly within a given parity. Lactating Holstein dairy cows (n = 704) were synchronized to receive timed artificial insemination (TAI) on Day 81 ± 3 of lactation by using the Double-Ovsynch protocol (GnRH-7d-PGF-3d-GnRH-7d-GnRH-7d-PGF-56h-GnRH-16h-TAI). At the time of the last injection of PGF, uterine diameter was determined at the greater curvature using ultrasound, uterine length was determined by rectal palpation, and uterine volume was calculated from these two measurements. Blood samples were also taken to measure progesterone to assure synchronization of all cows used in the final analysis (n = 616; primiparous, n = 289; multiparous, n = 327). Primiparous cows had greater percentage pregnant/AI (P/AI) compared to multiparous cows (49.8% vs. 39.1% at 67 days of pregnancy diagnosis, P = 0.009). Diameter, length, and volume of the uterus were larger in multiparous than in primiparous cows (P < 0.001). For multiparous cows, uterine diameter and volume were smaller in cows that became pregnant compared to cows that were not pregnant to the TAI with a similar tendency observed in primiparous cows. Logistic regression and quartile analysis also showed that as uterine volume increased, there was decreased P/AI in either primiparous or multiparous cows. Thus, there is a negative association between uterine size and fertility in lactating dairy cows with a larger uterus associated with reduced fertility, particularly for multiparous cows.

Effect of a second treatment with prostaglandin F2α during the Ovsynch protocol on luteolysis and pregnancy in dairy cows.

The main objective of this study was to evaluate the effect of a second treatment with prostaglandin F2α (PGF) during Ovsynch on regression of the corpus luteum (CL) and on fertility to the timed artificial insemination. Two experiments were performed. In both experiments, cows were randomized to receive (1) no additional treatments with PGF=1 PGF, or (2) a second PGF treatment at 24h after the first PGF treatment=2 PGF. The first experiment (n=344 synchronized lactating dairy cows that received artificial insemination at 81±3d in milk) used the Double-Ovsynch protocol for synchronizing ovulation. Blood samples were collected at the PGF and final GnRH treatments (72 and 16h before timed artificial insemination) during the breeding Ovsynch protocol, to determine CL regression in response to the protocol. Treatment with 2 PGF increased CL regression from 83.0% with 1 PGF to 97.0% with 2 PGF. The effect of 2 PGF on CL regression was observed in both primiparous and multiparous cows. Cows with lower (2.0 to 4.8ng/mL) versus greater (4.9 to 12.0ng/mL) circulating progesterone at the time of PGF had lower percentage of cows with complete CL regression after 1 PGF (66.7 vs. 88.1%) but not after 2 PGF (95.1 vs. 97.6%). Experiment 2 used 2,148 lactating dairy cows on 11 dairy farms in 4 different regions of the United States. Cows were synchronized with Ovsynch and received timed artificial insemination at 60±3d in milk. Cows that received 2 PGF had a tendency for increased pregnancies per artificial insemination (P/AI) compared with cows with 1 PGF (36.1 vs. 33.3%). This tendency for improvement in P/AI was observed in multiparous but not in primiparous cows. Combining data from the 2 experiments indicated a 9.45% relative increase in P/AI for cows receiving 2 compared with 1 PGF (37.6 vs. 34.4%) with the increase in P/AI observed in multiparous but not in primiparous cows. Thus, a second PGF treatment in Ovsynch-type protocols can increase pregnancy success by about 10%, primarily due to enhanced fertility in multiparous cows.

Use of a single injection of long-acting recombinant bovine FSH to superovulate Holstein heifers: a preliminary study.

Our objective was to compare several experimental preparations of a single injection of long-acting recombinant bovine FSH (rbFSH; types A and B) to a porcine pituitary-derived FSH (Folltropin) to superovulate Holstein dairy heifers. Nonlactating, nonpregnant virgin Holstein heifers (n = 56) aged 12 to 15 months were randomly assigned to one of four superstimulatory treatments. Beginning at a random stage of the estrous cycle, all follicles greater than 5 mm were aspirated. Thirty-six hours later, heifers received an intravaginal P4 device and superstimulatory treatments were initiated. Treatments were (1) 300 mg of pituitary-derived FSH (Folltropin) administered in eight decreasing doses over a period of 3.5 days; (2) a single injection of 50 µg of A-rbFSH; (3) a single injection of 100 µg of A-rbFSH; and (4) a single injection of 50 µg of B-rbFSH. All heifers received 25 mg PGF2α at 48 and 72 hours after the insertion of P4 device. At 84 hours after insertion, P4 devices were removed, and ovulation was induced 24 hours later with hCG (2500 IU). Heifers were inseminated at 12 and 24 hours after hCG treatment. The number of ovulatory follicles was greatest for heifers treated with Folltropin and B50-rbFSH, least for heifers treated with A50-rbFSH, and was intermediate for heifers treated with A100-rbFSH (25.7 ± 3.2, 18.9 ± 3.2, 5.9 ± 0.9, and 16.6 ± 3.1, respectively; P < 0.001). The number of corpora lutea was greatest for heifers treated with Folltropin, B50-rbFSH, and A100-rbFSH, and least for heifers treated with A50-rbFSH (19.1 ± 2.4, 16.1 ± 3.0, 15.9 ± 2.9, and 2.6 ± 0.9, respectively; P < 0.001). The number of good-quality embryos differed among treatments and was greatest for heifers treated with B50-rbFSH, Folltropin, and A100-rbFSH and least for heifers treated with A50-rbFSH (7.6 ± 2.4, 6.5 ± 1.7, 4.3 ± 1.5, and 0.8 ± 0.5, respectively; P < 0.001). In conclusion, a single injection of a preparation of long-acting rbFSH (either 100 µg of A-rbFSH or 50 µg of B-rbFSH but not 50 µg of A-rbFSH) produced similar superovulatory responses resulting in the production of good-quality embryos when compared with a pituitary-derived FSH preparation administered twice daily for 4 days. More studies using different types of cattle and different doses of rbFSH are needed to confirm the findings reported in this preliminary study.