Sperm are stored for extended periods of time in the epididymis, but upon ejaculation motility is increased and lifespan is decreased. The objective of this study was to identify differences in ...proteins between epididymis and ejaculated samples that are associated with longevity. Ejaculated semen was collected from mature Angus bulls (n = 9); bulls were slaughtered and epididymal semen was collected. Epididymal and ejaculated semen were centrifuged to separate sperm and fluid. Fluids were removed and sperm pellets were resuspended in a high ionic solution and vortexed to remove loosely attached proteins. Sperm samples were centrifuged, and the supernatant was removed; both fluid and sperm samples were snap frozen in liquid nitrogen and stored at −80 °C. Protein analysis was performed by LCMS/MS. A different group of yearling Angus cross bulls (n = 40) were used for sperm cultures. Ejaculated (n = 20) and epididymal (n = 20) semen were diluted and cultured in a commercial media at pH 5.8, 6.8 and 7.3, at 4 °C. Sperm were evaluated for motility and viability every 24 h until motility was lower than 20%. There was an effect of pH, time and pH by time interaction for motility and viability for both ejaculated and epididymal sperm (P ≤ 0.05). At 216 h of incubation epididymal sperm at pH 7.3 and ejaculated sperm at pH 6.8 reached motility below 20%. A total of 458 unique proteins were identified; 178, 298, 311, and 344 proteins were identified in ejaculated fluid, ejaculated sperm, epididymal fluid and epididymal sperm, respectively. There were 8, 24, 10, and 18 significant KEGG pathways (FDR <0.05) for ejaculated fluid, epididymal fluid, ejaculated sperm, and epididymal sperm, respectively. The metabolic pathway was identified as the most important KEGG pathway; glycolysis/gluconeogenesis, pentose phosphate, and glutathione metabolism pathways were significant among proteins only present in epididymal samples within the metabolic pathway. Other proteins identified that may be related to epididymal sperm's increased longevity were peroxidases and glutathione peroxidases for their antioxidant properties. In summary, energy metabolism in the epididymis appears to be more glycolytic compared to ejaculated and epididymis sperm have a larger number of antioxidants available which may be helping to maintain sperm in a quiescent state. Epididymal sperm remained viable (membrane integrity) longer than ejaculated sperm when cultured at the same pH.
Display omitted
•Epididymal sperm maintained viability longer than ejaculated sperm in culture.•Ejaculated sperm maintained motility longer when cultured at pH 6.8.•The metabolic pathway was identified as the most important for increased longevity.•Energy metabolism in the epididymis appears to be more glycolytic.•Increased numbers of antioxidants may help maintain sperm in a quiescent state.
Abstract
The focus of this review is maternal nutrition during the periconceptual period and offspring developmental outcomes in beef cattle, with an emphasis on the first 50 d of gestation, which ...represents the embryonic period. Animal agriculture in general, and specifically the beef cattle industry, currently faces immense challenges. The world needs to significantly increase its output of animal food products by 2050 and beyond to meet the food security and agricultural sustainability needs of the rapidly growing human population. Consequently, efficient and sustainable approaches to livestock production are essential. Maternal nutritional status is a major factor that leads to developmental programming of offspring outcomes. Developmental programming refers to the influence of pre-and postnatal factors, such as inappropriate maternal nutrition, that affect growth and development and result in long-term consequences for health and productivity of the offspring. In this review, we discuss recent studies in which we and others have addressed the questions, “Is development programmed periconceptually?” and, if so, “Does it matter practically to the offspring in production settings?” The reviewed studies have demonstrated that the periconceptual period is important not only for pregnancy establishment but also may be a critical period during which fetal, placental, and potentially postnatal development and function are programmed. The evidence for fetal and placental programming during the periconceptual period is strong and implies that research efforts to mitigate the negative and foster the positive benefits of developmental programming need to include robust investigative efforts during the periconceptual period to better understand the implications for life-long health and productivity.
Ovarian ultrasonography and measurement of circulating concentrations of anti-Müllerian hormone (AMH) have been used to estimate follicle number and predict fertility in mammalian females, but no ...study has evaluated follicle number and circulating concentrations of AMH in ewes known to differ in fertility. We tested the hypothesis that ewes that had failed to lamb (BARREN) in four consecutive annual breeding seasons of 21–35 d have fewer follicles and diminished circulating concentrations of AMH compared to closely matched ewes that consistently produced lambs (FERTILE) under the same breeding protocols. Once identified, BARREN ewes (n = 19) were paired by breed and sire to a FERTILE control ewe (n = 19) and reproductive tracts were recovered at necropsy. Visible antral follicles in both ovaries were counted and a representative cross section of one ovary was embedded for histological evaluation of pre-antral follicle numbers. Paired t-tests indicated that BARREN ewes had fewer antral follicles, fewer primordial follicles, and decreased circulating concentrations of AMH compared to FERTILE ewes (P ≤ 0.01), but there were ewes in each fertility classification that had ovarian phenotypes like the opposite fertility classification. The best technologies we have currently for estimating follicle numbers are ultrasonography and measurement of circulating concentrations of AMH, but no single technique is perfect for predicting fertility. A better understanding of the under-lying biological mechanisms linking AMH, folliculogenesis, and fertility is required to improve the use of measurements of follicle number for predicting fertility in livestock.
•Most infertile ewes had fewer follicles in their ovaries than fertile ewes.•Circulating anti-Müllerian hormone was decreased in most infertile ewes.•Predictors of follicle number could identify a large portion of the infertile ewes.
Embryo survival and pregnancy success is increased among animals that exhibit estrus prior to fixed time-artificial insemination, but there are no differences in conceptus survival to d16. The ...objective of this study was to determine effects of preovulatory estradiol on uterine transcriptomes, select trophectoderm (TE) transcripts, and uterine luminal fluid proteins. Beef cows/heifers were synchronized, artificially inseminated (d0), and grouped into either high (highE2) or low (lowE2) preovulatory estradiol. Uteri were flushed (d16); conceptuses and endometrial biopsies (n = 29) were collected. RNA sequencing was performed on endometrium. Real-time polymerase chain reaction (RT-PCR) was performed on TE (n = 21) RNA to measure relative abundance of IFNT, PTGS2, TM4SF1, C3, FGFR2, and GAPDH. Uterine fluid was analyzed using 2D Liquid Chromatography with tandem mass spectrometry-based Isobaric tags for relative and absolute quantitation (iTRAQ) method. RT-PCR data were analyzed using the MIXED procedure in SAS. There were no differences in messenger RNA (mRNA) abundances in TE, but there were 432 differentially expressed genes (253 downregulated, 179 upregulated) in highE2/conceptus versus lowE2/conceptus groups. There were also 48 differentially expressed proteins (19 upregulated, 29 downregulated); 6 of these were differentially expressed (FDR < 0.10) at the mRNA level. Similar pathways for mRNA and proteins included: calcium signaling, protein kinase A signaling, and corticotropin-releasing hormone signaling. These differences in uterine function may be preparing the conceptus for improved likelihood of survival after d16 among highE2 animals. Summary sentence Preovulatory estradiol did not impact conceptus survival to d16; however, it did influence uterine gene/protein expressions related to adhesion, endometrial remodeling, metabolism, and immune regulation, which may explain improved pregnancy success.
Protease-activated receptor 4 (PAR4) (gene F2RL3) harbors a functional dimorphism, rs773902 A/G (encoding Thr120/Ala120, respectively) and is associated with greater platelet aggregation. The A ...allele frequency is more common in Black individuals, and Black individuals have a higher incidence of ischemic stroke than White individuals. However, it is not known whether the A allele is responsible for worse stroke outcomes. To directly test the in vivo effect of this variant on stroke, we generated mice in which F2rl3 was replaced by F2RL3, thereby expressing human PAR4 (hPAR4) with either Thr120 or Ala120. Compared with hPAR4 Ala120 mice, hPAR4 Thr120 mice had worse stroke outcomes, mediated in part by enhanced platelet activation and platelet-neutrophil interactions. Analyses of 7,620 Black subjects with 487 incident ischemic strokes demonstrated the AA genotype was a risk for incident ischemic stroke and worse functional outcomes. In humanized mice, ticagrelor with or without aspirin improved stroke outcomes in hPAR4 Ala120 mice, but not in hPAR4 Thr120 mice. P selectin blockade improved stroke outcomes and reduced platelet-neutrophil interactions in hPAR4 Thr120 mice. Our results may explain some of the racial disparity in stroke and support the need for studies of nonstandard antiplatelet therapies for patients expressing PAR4 Thr120.
Lifetime productivity and longevity are greater in beef cows that give birth early in their first calving season. The ability of heifers to conceive early in the breeding season is traditionally ...thought to be a function of pubertal status; however, recent evidence suggests that antral follicle count is positively associated with calving day in pubertal beef heifers. Additionally, there is evidence to suggest that the total number of ovarian follicles may alter response to hormonal synchronization protocols. The objectives of this study were to confirm the beneficial influence of antral follicle count on calving day in beef heifers and to determine whether response to PGF2α is associated with differences in antral follicle counts. A 2 × 2 factorial experiment was designed to investigate the interaction between calving period (early vs. late) and PGF2α (control vs. PGF) on calving day and antral follicle count in yearling beef heifers (n = 95). As intended, calving day was less in the early calving period compared to the late calving period (P < 0.01). There were no differences in calving day in response to treatment with PGF2α (P > 0.05). There was a significant interaction between calving period and PGF2α on age at first calving (P < 0.01), such that heifers treated with PGF2α that gave birth early were younger than heifers treated with saline that gave birth early. Calf weaning weights were greater in the early calving group than in the late calving group (P < 0.01). Heifers that gave birth in the early calving group possessed more antral follicles at prebreeding ultrasonographic examination than heifers that gave birth in the late calving group (P = 0.05). These findings confirm that antral follicle counts are associated with calving day in pubertal beef heifers. The use of antral follicle counts as a prebreeding phenotype provides additional utility to reproductive tract scoring for commercial production because of its association with calving day. As a prebreeding ovarian phenotype, antral follicle counts may hold additional applicability for organic beef producers looking to reduce the length of their calving season without increasing the total number of replacement heifers retained.
The uterine environment must provide sufficient endocrine conditions and nutrients for pregnancy maintenance and conceptus survival. The objective of this study was to determine the effects of ...preovulatory estradiol and conceptus presence on uterine transcripts and uterine luminal fluid (ULF) proteins. Beef cows/heifers were synchronized and artificially inseminated (d 0). Uteri were flushed (d 16); conceptuses and endometrial biopsies were collected. Total cellular RNA was extracted from endometrium for RNA sequencing and RT-PCR validation. There were two independent ULF pools made for each of the following groups: highE2/conceptus, highE2/noconceptus, lowE2/conceptus, and lowE2/noconceptus that were analyzed using the 2D LC-MS/MS based iTRAQ method. There were 64 differentially expressed genes (DEGs) and 77 differentially expressed proteins (DEPs) in common among the highE2/conceptus vs highE2/noconceptus and lowE2/conceptus vs lowE2/noconceptus groups. In summary, the interaction between preovulatory estradiol and the conceptus induces the expression of genes, proteins, and pathways necessary for pregnancy.
•There were 64 DEGs in common among the highE2/conceptus vs highE2/noconceptus groups and lowE2/conceptus vs lowE2/noconceptus groups.•There were 77 DEPs in common among highE2/conceptus vs highE2/noconceptus and lowE2/conceptus vs lowE2/noconceptus.•The interaction between preovulatory estradiol and conceptus presence influences uterine gene and protein expression.
A naturally occurring bovine model with excess follicular fluid androstenedione (High A4), reduced fertility, and polycystic ovary syndrome (PCOS)-like characteristics has been identified. We ...hypothesized High A4 granulosa cells (GCs) would exhibit altered cell proliferation and/or steroidogenesis. Microarrays of Control and High A4 GCs combined with Ingenuity Pathway Analysis indicated that High A4 GCs had cell cycle inhibition and increased expression of microRNAs that inhibit cell cycle genes. Granulosa cell culture confirmed that A4 treatment decreased GC proliferation, increased anti-Müllerian hormone, and increased mRNA for CTNNBIP1. Increased CTNNBIP1 prevents CTNNB1 from interacting with members of the WNT signaling pathway thereby inhibiting the cell cycle. Expression of CYP17A1 was upregulated in High A4 GCs presumably due to reduced FOS mRNA expression compared to Control granulosa cells. Furthermore, comparisons of High A4 GC with thecal and luteal cell transcriptomes indicated an altered cellular identity and function contributing to a PCOS-like phenotype.
Display omitted
•Bovine granulosa cell gene expression in excess androgen microenvironment.•Androgen excess causes cell cycle arrest and increased AMH expression in granulosa cells.•Increased AMH contributs to increased CTNNBIP1 which reduces granulosa cell proliferation.•Reduced FOS abundance in excess androgen granulosa cell microenvironment correlates with increased CYP17A1.•Androgen excess microenvironment correlates with increased miRNA which target cell cycle genes.
The objective of this study was to determine the dose of folate and vitamin B12 in beef heifers fed rumen protected methionine and choline required to maintain increased B12 levels and intermediates ...of the methionine-folate cycle in circulation. Angus heifers (n = 30; BW = 392.6 ± 12.6 kg) were individually fed and assigned to one of five treatments: 0XNEG: Total mixed ration (TMR) and saline injections at day 0 and 7 of the estrous cycle, 0XPOS: TMR, rumen protected methionine (MET) fed at 0.08% of the diet DM, rumen protected choline (CHOL) fed at 60 g/d, and saline injections at day 0 and 7, 0.5X: TMR, MET, CHOL, 5 mg B12, and 80 mg folate at day 0 and 7, 1X: TMR, MET CHOL, 10 mg vitamin B12, and 160 mg folate at day 0 and 7, and 2X: TMR, MET, CHOL, 20 mg B12, and 320 mg folate at day 0 and 7. All heifers were estrus synchronized but not bred, and blood was collected on day 0, 2, 5, 7, 9, 12, and 14 of a synchronized estrous cycle. Heifers were slaughtered on day 14 of the estrous cycle for liver collection. Serum B12 concentrations were greater in the 0.5X, 1X, and 2X, compared with 0XNEG and 0XPOS on all days after treatment initiation (P < 0.0001). Serum folate concentrations were greater for the 2X treatment at day 5, 7, and 9 of the cycle compared with all other treatments (P ≤ 0.05). There were no differences (P ≥ 0.19) in hepatic methionine-cycle or choline analyte concentrations by treatment. Concentrations of hepatic folate cycle intermediates were always greater (P ≤ 0.04) in the 2X treatment compared with the 0XNEG and 0XPOS heifers. Serum methionine was greater (P = 0.04) in the 0.5X and 2X heifers compared with 0XNEG, and S-adenosylhomocysteine (SAH) tended (P = 0.06) to be greater in the 0.5X heifers and the S-adenosylmethionine (SAM):SAH ratio was decreased (P = 0.05) in the 0.5X treatment compared with the 0XNEG, 0XPOS, and 2X heifers. The hepatic transcript abundance of MAT2A and MAT2B were decreased (P ≤ 0.02) in the 0.5X heifers compared with the 0XNEG, 0XPOS, and 2X heifers. These data support that beef heifers fed rumen protected methionine and choline require 20 mg B12 and 320 mg folate once weekly to maintain increased concentrations of B12 and folate in serum. Furthermore, these data demonstrate that not all supplementation levels are equal in providing positive responses, and that some levels, such as the 0.5X, may result in a stoichiometric imbalance in the one-carbon metabolism pathway that results in a decreased SAM:SAH ratio.
In ruminants, prostaglandin F2alpha (PGF2α)-mediated luteolysis is essential prior to estrous cycle resumption, and is a target for improving fertility. To deduce early PGF2α-provoked changes in the ...corpus luteum a short time-course (0.5–4 h) was performed on cows at midcycle. A microarray-determined transcriptome was established and examined by bioinformatic pathway analysis. Classic PGF2α effects were evident by changes in early response genes (FOS, JUN, ATF3) and prediction of active pathways (PKC, MAPK). Several cytokine transcripts were elevated and NF-κB and STAT activation were predicted by pathway analysis. Self-organizing map analysis grouped differentially expressed transcripts into ten mRNA expression patterns indicative of temporal signaling cascades. Comparison with two analogous datasets revealed a conserved group of 124 transcripts similarly altered by PGF2α treatment, which both, directly and indirectly, indicated cytokine activation. Elevated levels of cytokine transcripts after PGF2α and predicted activation of cytokine pathways implicate inflammatory reactions early in PGF2α-mediated luteolysis.
Display omitted
•Analysis of the bovine luteal transcriptome at 0.5–4 h post-PGF2α treatment.•Transcriptomics and bioinformatics predict classical PGF2α and cytokine signaling.•Reduced cholesterol availability may contribute to early decreases in progesterone.•PGF2α rapidly changes cytokine and cytokine targets expression at < 4 h.•Reduced progesterone and increased cytokine signals may prime the CL for luteolysis.
PDF
