Sire modulates developmental kinetics and transcriptome of the bovine embryo.

In brief: The first week of gestation is a period of major pregnancy loss in cattle, this study reveals that the male plays a key role in regulating embryonic development during this time. Abstract: The impact of sire on preimplantation embryonic development in cattle remains poorly understood. This study evaluated differences in embryos produced in vitro from sires with varying capacities to produce blastocysts. Sires classified as high (HP) and low performing (LP) based on their ability to produce embryos were used to better understand how sire regulates embryonic development. By monitoring development, it was determined that the most common arrest stage was the five- to six-cell stage. Embryos (four to six cells) from HP and LP sires were then analyzed for autophagic activity, where embryos for LP sires exhibited increased autophagy than HP-derived embryos. Transcriptome analysis of four-cell embryos found that embryos from LP sires might have issues in sperm mitochondrial clearance, histone retention, and DNA damage, while HP sires had increased expression of genes involved in transcription, chromosome segregation, and cell division. In conclusion, LP sires had an increased proportion of embryos arresting at the five- to six-cell stage, and these embryos had higher rates of cellular stress due to paternal contributions from the spermatozoon.

Disruption of CSF2RA in the bovine preimplantation embryo reduces development and affects embryonic gene expression in utero.

The hypothesis that CSF2 plays a role in the preimplantation development of the bovine embryo was tested by evaluating consequences of inactivation of CSF2RA (the functional receptor in the embryo) for development of embryos in utero. CRISPR/Cas9 was used to alter sequences on exon 5 and intron 5 of CSF2RA, Control embryos were injected with Cas9 mRNA only. Embryos > 16 cells at day 5 after insemination were transferred to synchronized recipient females in groups of 7 to 24. Embryos were flushed from the uterus two days later. The proportion of recovered embryos that developed to the blastocyst stage was lower for knockout embryos (39%) than for control embryos (63%). RNA sequencing of individual morulae and blastocysts indicated a total of 27 (morula) or 15 (blastocyst) differentially-expressed genes (false discovery rate <0.05). Gene set enrichment analysis indicated that the knockout affected genes playing roles in several functions including cell signaling and glycosylation. It was concluded that signaling through CSF2RA is not obligatory for development of the bovine preimplantation embryo to the blastocyst stage but that CSF2 signaling does enhance the likelihood that the embryo can become a blastocyst and result in specific changes in gene expression.

Uterine lumen fluid is metabolically semi-autonomous.

Uterine lumen fluid (ULF) is central to successful pregnancy establishment and maintenance, and impacts offspring wellbeing into adulthood. The current dogma is that ULF composition is primarily governed by endometrial glandular epithelial cell secretions and influenced by progesterone. To investigate the hypothesis that ULF is metabolically semi-autonomous, ULF was obtained from cyclic heifers, and aliquots incubated for various durations prior to analysis by untargeted semi-quantitative metabolomic profiling. Metabolite flux was observed in these ULF isolates, supporting the idea that the biochemical makeup of ULF is semi-autonomously dynamic due to enzyme activities. Subsequent integrative analyses of these, and existing, data predict the specific reactions underpinning this phenomenon. These findings enhance our understanding of the mechanisms leading to pregnancy establishment, with implications for improving fertility and pregnancy outcomes in domestic animals as well as women.

Downregulated luteolytic pathways in the transcriptome of early pregnancy bovine corpus luteum are mimicked by interferon-tau in vitro.

BACKGROUND: Maintenance of the corpus luteum (CL) beyond the time of luteolysis is essential for establishing pregnancy. Identifying the distinct features of early pregnancy CL remains unresolved, hence we analyzed here the transcriptome of CL on day 18 pregnant (P) and non-pregnant (NP) cows using RNA-Seq. CL of P cows expressed ISGs, verifying exposure to the pregnancy recognition signal, interferon-tau (IFNT), whereas the CL of NP cows had elevated luteal progesterone levels, implying that luteolysis had not yet commenced. RESULTS: The DEGs, IPA, and metascape canonical pathways, along with GSEA analysis, differed markedly in the CL of P cows from those of NP cows, at the same day of the cycle. Both metascape and IPA identified similar significantly enriched pathways such as interferon alpha/beta, sonic hedgehog pathway, TNFA, EDN1, TGFB1, and PDGF. However, type-1 interferon and sonic hedgehog pathways were positively enriched whereas most of the enriched pathways were downregulated in the P compared to NP samples. Thirty-four % of these pathways are known to be elevated by PGF2A during luteolysis. Notably, selective DEGs in luteinized granulosa cells were modulated by IFNT in vitro in a similar manner to their regulation in the CL of P cows. CONCLUSION: This study unraveled the unique transcriptomic signature of the IFNT-exposed, early pregnancy CL, highlighting the abundance of downregulated pathways known to be otherwise induced during luteolysis. These and IFNT-regulated in vitro pregnancy-specific DEGs suggest that IFNT contributes to the characteristics and maintenance of early pregnancy CL.

Interactions of circulating estradiol and progesterone on changes in endometrial area and pituitary responsiveness to GnRH†.

Changes in circulating progesterone (P4) and estradiol (E2) during proestrus produce dynamic changes in endometrial function and pituitary release of gonadotropins. Independent and combined effects of P4 and E2 on endometrium and pituitary were evaluated. In a preliminary study, an exogenous hormone model of proestrus was created by removal of corpus luteum and follicles ≥5 mm followed by gradual removal of intravaginal P4 implants during 18 h and treatment with increasing doses of estradiol benzoate during 48 h to mimic proestrus using high E2 (n = 9) or low E2 (n = 9). Decreased P4, increased E2, and increased endometrial area (EA) simulated proestrus in high-E2 cows and this was used subsequently. The main experiment used a 2 × 2 factorial design with: high E2 and low P4 (n = 11); high E2 and high P4 (n = 11); low E2 and high P4 (n = 11); low E2 and low P4 (n = 10). At 48 h, gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) and follicle stimulating hormone (FSH) release was determined. Variables were analyzed using PROCMIXED of Statistical Analysis System. The EA increased dramatically during 48 h only in high-E2 and low-P4 cows. For FSH, high-E2 cows had greater area under the curve (AUC) and FSH peak after GnRH than low E2, with mild negative effects of high P4. For LH, concentration at peak and AUC were 2-fold greater in high E2 compared to low-E2 groups, with low P4 also 2-fold greater than high-P4 groups. Thus, maximal changes in uterus and pituitary during proestrus depend on both low P4 and high E2, but different physiologic responses are regulated differently by E2 and P4. Changes in endometrium depend on low P4 and high E2, whereas GnRH-induced FSH secretion primarily depends on high E2, and GnRH-induced LH secretion is independently increased by high E2 or reduced by high P4.

Progesterone-based timed AI protocols for Bos indicus cattle I: Evaluation of ovarian function.

Three experiments evaluated ovarian dynamics and circulating progesterone (P4) during P4-based protocols initiated with GnRH, estradiol benzoate (EB), or no additional treatment in Nelore (Bos indicus) cattle. In Exp 1 (n = 59 cows), a 5-d P4-only protocol (P-5d; D0: P4 implant alone (1g); D5: P4 removal, 0.5 mg estradiol cypionate [EC], 0.526 mg cloprostenol [PGF], and 300 IU equine chorionic gonadotropin [eCG]; D7: 8.4 µg buserelin acetate [GnRH]) was compared to a 9d protocol initiated with EB (EB-9d; D0: 2 mg EB + P4; D9: P4 removal + EC + PGF + eCG), and to a 7d GnRH protocol (G-7d; D0: 16.8 µg GnRH + P4; D6: PGF + eCG; D7: P4 removal + PGF; D9: GnRH). Exp 2 (n = 55 cows) compared G-7d and EB-7d protocols (similar to EB-9d, but D9 treatments were done on D7). Exp 3 (n = 64 heifers) compared EB-7d, G-7d, and P-5d protocols. For all experiments, daily ovarian ultrasonography was done from D0 until 4d after implant withdrawal and blood samples were collected at D0 and first PGF. Follicle dynamics were determined for each individual animal, analyzed within individual experiments, and afterwards combined to determine overall effects of treatments. The protocol that began with GnRH, G-7d, had greater ovulation rate after D0 with subsequently greater number of CL and circulating P4 at time of PGF (52.8%, 1.0 ± 0.1 CL, 4.0 ± 0.4 ng/mL) than for EB protocols (12.1%, 0.4 ± 0.05 CL, 2.0 ± 0.2 ng/mL), or P-5d (2.5%, 0.6 ± 0.09 CL, 2.6 ± 0.3 ng/mL). The G-7d and EB protocols had synchronized follicle wave emergence in 92.1% of animals but with distinct patterns. For the G-7d group, wave emergence occurred earlier in ovulating than non-ovulating animals (1.4 ± 0.2 d vs 2.5 ± 0.4 d). By comparison, most animals in EB-7d or EB-9d (80.3%) displayed atresia of the dominant follicle, followed by wave emergence 2-3 d after EB treatment. In contrast, P-5d protocol synchronized wave emergence in only 30.0% of cows. Nevertheless, no differences among treatments were detected for ovulation at end of the protocol (85.7%). In conclusion, the P-5d protocol did not synchronize follicle wave emergence but produced similar final ovulation, whereas, GnRH and EB protocols had follicle dynamics synchronized by distinct mechanisms that produced differences in CL number and P4 at the time of PGF treatment but similar final ovulation. Based on ovarian function, each of these synchronization methods are promising for use in FTAI, although fertility still needs to be evaluated.

Progesterone-based timed AI protocols for Bos indicus cattle II: Reproductive outcomes of either EB or GnRH-type protocol, using or not GnRH at AI.

The aim of these experiments was to study ovarian dynamics and fertility of Bos indicus beef cattle submitted to 7-d progesterone (P4)-based fixed-time AI (FTAI) protocols using different hormonal treatments. In Exp. 1, 2 yr old Nelore heifers (n = 973) were randomly assigned to one of four treatments: EB-0 (estradiol benzoate, EB on D0 and no GnRH at AI), EB-G (EB on D0 and GnRH at AI), G-0 (GnRH on D0 and no GnRH at AI), or G-G (GnRH on D0 and at AI). On D0, heifers received an intravaginal P4 implant (0.5 g) for 7 d and EB (1.5 mg) or GnRH (16.8 µg). On D7, the P4 implant was withdrawn and heifers received cloprostenol (PGF; 0.5 mg) and estradiol cypionate (EC, 0.5 mg). Heifers in G groups also received PGF and eCG (200 IU) on D6, whereas EB heifers received eCG on D7. At FTAI on D9, only EB-G and G-G groups received GnRH (8.4 µg). In Exp. 2, Nelore cows (n = 804) received the same treatments (EB-0, EB-G, G-0, or G-G) using a 1.0 g P4 implant, 2.0 mg EB, and 300 IU eCG. Effects were considered significant when P ≤ 0.05. After treatment on D0, G had more ovulations than EB in heifers (60.3 [287/476] vs. 12.7% [63/497]) and cows (73.7 [83/112] vs. 24.4% [28/113]). Luteolysis after D0 was greater in EB than G in heifers (39.2 [159/406] vs. 20.0% [77/385]) and cows (25.5 [14/55] vs. 1.6% [1/64]). Heifers in G had larger follicles (mm) than EB on D7 (10.3 ± 0.2 vs. 9.2 ± 0.2) and at AI (11.9 ± 0.2 vs. 11.3 ± 0.2). Cows had larger follicles in G than EB on D7 (11.0 ± 0.3 vs. 9.9 ± 0.3) but not at AI. More estrus was observed in G than EB for heifers (80.3 [382/476] vs. 69.6% [346/497]) and cows (67.6 [270/400] vs. 56.2% [227/404]). There was no interaction between D0 and D9 treatments on pregnancy per AI (P/AI) in heifers (EB-0: 56.7 [139/245], EB-G: 53.6 [135/252], G-0: 52.6 [127/241], and G-G: 57.5% [135/235]). However, cows from EB-G had greater P/AI than EB-0 (69.5 [142/204] vs. 60.2% [120/200]), whereas P/AI for G-0 (62.7% [127/203]) was similar to G-G (60.9% [120/197]). In heifers, there was no interaction of GnRH at AI with estrus, however, cows that did not display estrus had greater P/AI if they received GnRH at AI (GnRH = 59.1 [91/154] vs. No GnRH = 48.2% [78/162]). Thus, protocols initiated with EB or GnRH for Bos indicus heifers and cows had differing ovarian dynamics but similar overall fertility, enabling their use in reproductive management programs. Treatment with GnRH at time of AI increased fertility in some instances in Bos indicus cows but not in heifers.

Oxytocin-induced prostaglandin F2-alpha release is low in early bovine pregnancy but increases during the second month of pregnancy†.

Circulating prostaglandin F2α metabolite (PGFM) after an oxytocin challenge was evaluated throughout the first 2 months of pregnancy in lactating Holstein cows. On day 11, 18, and 25 after artificial insemination (AI), and on days 32, 39, 46, 53, and 60 of pregnancy, cows were challenged with 50 IU oxytocin, i.m. Blood was collected before (0 min), 30, 60, 90, and 120 min after oxytocin for plasma PGFM concentrations. Ultrasound evaluations were performed for pregnancy diagnosis on day 32-60 post-AI. Nonpregnant (NP) cows on day 18 were designated by a lack of interferon-stimulated genes in peripheral blood leukocytes and Pregnant (P) based on day 32 ultrasound. On day 11, P and NP were similar with low PGFM and no effect of oxytocin on PGFM. On day 18, oxytocin increased PGFM (3-fold) in NP with little change in P cows. Comparing only P cows from day 11 to 60, basal circulating PGFM increased as pregnancy progressed, with day 11 and 18, lower than all days from day 25 to 60 of pregnancy. Oxytocin-induced PGFM in P cows on day 25 was greater than P cows on day 18 (2.9-fold). However, oxytocin-induced PGFM was lower on day 25 compared to day 53 and 60, with intermediate values on day 32, 39, and 46 of pregnancy. Thus, the corpus luteum (CL) of early pregnancy (day 11, 18) is maintained by suppression of PGF, as reflected by suppressed PGFM in this study. However, during the second month of pregnancy, uterine PGF secretion was not suppressed since basal PGFM and oxytocin-induced PGFM secretion were elevated. Apparently, mechanisms other than suppression of oxytocin receptors maintain CL after day 25 of pregnancy.

Interferon-Tau Exerts Direct Prosurvival and Antiapoptotic Actions in Luteinized Bovine Granulosa Cells.

Interferon-tau (IFNT), serves as a signal to maintain the corpus luteum (CL) during early pregnancy in domestic ruminants. We investigated here whether IFNT directly affects the function of luteinized bovine granulosa cells (LGCs), a model for large-luteal cells. Recombinant ovine IFNT (roIFNT) induced the IFN-stimulated genes (ISGs; MX2, ISG15, and OAS1Y). IFNT induced a rapid and transient (15-45 min) phosphorylation of STAT1, while total STAT1 protein was higher only after 24 h. IFNT treatment elevated viable LGCs numbers and decreased dead/apoptotic cell counts. Consistent with these effects on cell viability, IFNT upregulated cell survival proteins (MCL1, BCL-xL, and XIAP) and reduced the levels of gamma-H2AX, cleaved caspase-3, and thrombospondin-2 (THBS2) implicated in apoptosis. Notably, IFNT reversed the actions of THBS1 on cell viability, XIAP, and cleaved caspase-3. Furthermore, roIFNT stimulated proangiogenic genes, including FGF2, PDGFB, and PDGFAR. Corroborating the in vitro observations, CL collected from day 18 pregnant cows comprised higher ISGs together with elevated FGF2, PDGFB, and XIAP, compared with CL derived from day 18 cyclic cows. This study reveals that IFNT activates diverse pathways in LGCs, promoting survival and blood vessel stabilization while suppressing cell death signals. These mechanisms might contribute to CL maintenance during early pregnancy.

Effect of different chorionic gonadotropins on final growth of the dominant follicle in Bos indicus cows.

The aim of this study was to evaluate the effect of eCG or hCG on the final growth of the dominant follicle in Nelore (Bos indicus) cows submitted to fixed-time AI (FTAI). Eighty-four lactating cows with body condition score (BCS) of 2.9 (range 1-5) were used. At a random day of the estrous cycle (D0) cows received 2 mg estradiol benzoate and a reused intravaginal progesterone device (1.9 g). At D8, when the device was removed, 0.5 mg cloprostenol and 1 mg estradiol cypionate was given i.m., and cows were randomly assigned to receive on D8 one of the following treatments: Control (no treatment; n = 17), eCG (300 IU i.m.; n = 17), hCG 300 (300 IU i.m.; n = 18), hCG 200 IM (200 IU i.m.; n = 16) and hCG 200 SC (200 IU s.c.; n = 16). On the same day and 2 days later, cows were subjected to ovarian ultrasonography to evaluate the diameter of the largest follicle and to calculate follicular growth rate (D8 to D10). No differences were observed for the diameter of the largest follicle on D8 (P = 0.3) or D10 (P = 0.4) among treatments. However, the growth rate of the dominant follicle between D8 and D10 was greater for the groups eCG and hCG 300 and there were no differences between the other treatments (Control = 1.1 mm/day; eCG = 1.8 mm/day; hCG 300 = 1.8 mm/day; hCG 200 IM = 1.3 mm/day; hCG 200 SC = 1.3 mm/day; P = 0.02). In addition, more cows from the Group hCG 300 presented premature ovulation (44.4%) than cows from Control (5.8%), eCG (0%), or hCG 200 IM (12.5%), but did not differ from Group hCG 200 SC (18.7%). Regardless of treatment, the size of the largest follicle on D8 was different between cows that presented premature ovulation vs. cows that did not ovulate prematurely (11.3 mm vs. 9.9 mm, respectively; P = 0.01). Treatment with different hCG doses on D8 of a FTAI protocol failed to produce similar effects compared to eCG in terms of final follicular growth support and greater ovulatory follicle size. In addition, the groups hCG 300 and hCG 200 SC induced premature ovulation in a greater portion of cows. Thus, a single administration of hCG on D8 does not appear to be a reliable alternative to eCG treatment in FTAI protocols.

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.

Equine chorionic gonadotropin increases fertility of grazing dairy cows that receive fixed-time artificial insemination in the early but not later postpartum period.

This study evaluated effects of equine chorionic gonadotropin (eCG) on fertility of 679 crossbred (Bos taurus x Bos indicus) lactating grazing cows synchronized for fixed-time AI (FTAI). At a random day of the estrous cycle cows received an intravaginal progesterone (P4) implant, 2 mg estradiol benzoate (EB) and 100 µg gonadorelin (D0-AM). On D7-AM, cows received 0.5 mg sodium cloprostenol and were randomly assigned into two treatments: eCG (n = 340; 400 IU eCG on D7), or Control (n = 339; no eCG). On D8-PM, P4 implants were removed and cows received 0.5 mg sodium cloprostenol and 1 mg EB. Insemination was performed on D10-AM. Pregnancy was diagnosed 30 and 60 d after AI. Treatment with eCG tended to increase pregnancy per AI (P/AI) compared to Control at 30 (37.8 vs. 30.2%; P = 0.06) and 60 (31.9 vs. 25.1%; P = 0.08) d. Pregnancy loss and twinning did not differ between groups. Treatment with eCG increased (P < 0.05) P/AI at 30 (39.0 vs. 25.2%) and 60 (32.8 vs. 21.3%) d for cows inseminated at ≤ 70 d in milk (DIM) but had no effect in cows receiving AI after 70 DIM. Thus, eCG on D7 of a FTAI protocol increased fertility of crossbred dairy cows inseminated in the early postpartum period.