Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, in vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo versus in vitro and in vivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between in vivo and cloned conceptuses, while only 6 miRNAs were different between in vivo and in vitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy.

The metabolomic composition of the oviductal fluid is controlled by the periovulatory hormonal context in Bos indicus cows.

In cattle, oviductal function is controlled by the ovarian sex-steroids estradiol and progesterone. Here, we tested the hypothesis that the exposure to contrasting sex-steroid milieus differentially impacts the oviductal fluid composition. Estrous cycles of non-lactating, multiparous Nelore cows were pre-synchronized and then synchronized with a protocol designed two induce ovulation of large (LF group) or small (SF group) follciles. Larger preovulatory follicle (day 0) and corpora lutea (day 4) and greater estradiol (day 0) and progesterone (day 4) concentrations were observed in the LF group. Four days after induced ovulation, oviductal fluid was collected post-mortem. Quantitative mass spectrometry was used to determine the concentration of amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, lysophosphatidylcholines, sphingomyelins, hexoses, prostaglandins and related compounds. Multivariate analyses (OPLS-DA) were conducted to compare the metabolomic signatures of oviductal fluids. Correlation network analysis was conducted to measure the strength and hierarchy of associations among metabolites. Of the 205 metabolites quantified, 171 were detected in at least 50% of the samples and were included in further data analysis. After OPLS-DA analysis, samples of the LF and SF were divided clearly into two non-overlapping clusters. Twenty metabolites had different or tended to have different concentrations in the oviductal fluid when comparing groups. Seven of these 20 analytes had greater concentrations in LF cows. Moreover, total sum of biogenic amines, phosphatidylcholines, and prostaglandins were higher in the SF group. The correlation network showed that the LF group metabolites' concentrations were highly intercorrelated, which was not observed in the SF group. We concluded that the periovulatory endocrine milieu regulates the composition of the oviductal fluid.

Provision of choline chloride to the bovine preimplantation embryo alters postnatal body size and DNA methylation†.

Choline is a vital micronutrient. In this study, we aimed to confirm, and expand on previous findings, how choline impacts embryos from the first 7 days of development to affect postnatal phenotype. Bos indicus embryos were cultured in a choline-free medium (termed vehicle) or medium supplemented with 1.8 mM choline. Blastocyst-stage embryos were transferred into crossbred recipients. Once born, calves were evaluated at birth, 94 days, 178 days, and at weaning (average age = 239 days). Following weaning, all calves were enrolled into a feed efficiency trial before being separated by sex, with males being slaughtered at ~580 days of age. Results confirm that exposure of 1.8 mM choline chloride during the first 7 days of development alters postnatal characteristics of the resultant calves. Calves of both sexes from choline-treated embryos were consistently heavier through weaning and males had heavier testes at 3 months of age. There were sex-dependent alterations in DNA methylation in whole blood caused by choline treatment. After weaning, feed efficiency was affected by an interaction with sex, with choline calves being more efficient for females and less efficient for males. Calves from choline-treated embryos were heavier, or tended to be heavier, than calves from vehicle embryos at all observations after weaning. Carcass weight was heavier for choline calves and the cross-sectional area of the longissimus thoracis muscle was increased by choline.

Supplementation with long-acting injectable progesterone 3 days after TAI impaired luteal function in buffaloes.

Three experiments were conducted to evaluate the effects of long-acting injectable progesterone (iP4) in buffalo cows. In Experiment 1, ovariectomized buffaloes received 300 mg (iP300) or 600 mg (iP600) of iP4, and serum P4 concentrations were evaluated. In experiment 2, three groups were compared: control or administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after timed artificial insemination (TAI). On day 16, reproductive tract was recovered for conceptus, endometrium, and corpus luteum (CL) analysis. In experiment 3, pregnancy per AI (P/TAI) and proportion of pregnancy losses were evaluated after administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after TAI in lactating buffaloes. In experiment 1, serum P4 concentrations remained over 1 ng/mL for ~ 3 days in both groups. The 300 mg dose was used in subsequent experiments. In experiment 2, CL weight and endometrial glands density were decreased, and conceptus length was increased in iP4-D3 compared to control and to iP4-D6 (P < 0.05). Transcript abundance of Prostaglandin F Receptor (FP) and ISG15 in CL and of ISG15 and MX1 in endometrium was greater in iP4-D3 when compared to control and to iP4-D6 (P < 0.05). In experiment 3, there was no difference among experimental groups for P/TAI at D30 and pregnancy losses (P > 0.1); however, iP4-D3 presented a lower P/TAI at day 60 (41.7%) when compared to control (56.8%) and iP4-D6 (57.7%; P = 0.07). In conclusion, administration iP4 at 3 days after TAI affects CL development and consequently decreases final pregnancy outcome in buffaloes.

Induced endometrial inflammation compromises conceptus development in dairy cattle†.

Endometrial inflammation is associated with reduced pregnancy per artificial insemination (AI) and increased pregnancy loss in cows. It was hypothesized that induced endometritis alters histotroph composition and induces inflammatory signatures on conceptus that compromise development. In Experiment 1, lactating cows were assigned to control (CON; n = 23) or to an intrauterine infusion of Escherichia coli and Trueperella pyogenes (ENDO; n = 34) to induce endometritis. Cows received AI 26 days after treatment, and the uterine fluid and conceptuses were collected on day 16 after AI. In Experiment 2, Holstein heifers were assigned to CON (n = 14) or ENDO (n = 14). An embryo was transferred on day 7 of the estrous cycle, and uterine fluid and conceptuses were recovered on day 16. Composition of histotroph and trophoblast and embryonic disc gene expression were assessed. Bacterial-induced endometritis in lactating cows altered histotroph composition and pathways linked to phospholipid synthesis, cellular energy production, and the Warburg effect. Also, ENDO reduced conceptus length in cows and altered expression of genes involved in pathogen recognition, nutrient uptake, cell growth, choline metabolism, and conceptus signaling needed for maternal recognition of pregnancy. The impact of ENDO was lesser on conceptuses from heifers receiving embryo transfer; however, the affected genes and associated pathways involved restricted growth and increased immune response similar to the observed responses to ENDO in conceptuses from lactating cows. Bacterial-induced endometrial inflammation altered histotroph composition, reduced conceptus growth, and caused embryonic cells to activate survival rather than anabolic pathways that could compromise development.

Pre-estrus progesterone does not affect post-estrus luminal metabolome in cross-bred beef cows.

In brief: The concentration of progesterone through the estrous cycle modulates uterine function to affect the luminal metabolome. This paper reports that the dynamic changes in the bovine uterine luminal metabolome during diestrus are independent of the concentration of progesterone in the previous cycle. Abstract: In cattle, the concentration of sex steroids modulates uterine function, which is reflected in the composition of the luminal metabolome. Ultimately, the uterine luminal metabolome influences embryonic growth and development. Our objectives were (i) to compare the luminal metabolome 4, 7, and 14 days after estrus of cows that were exposed to greater (HP4; n = 16) vs lower (LP4; n = 24) concentrations of progesterone before displaying estrus and ovulating spontaneously and (ii) to identify changes in the luminal concentration of metabolites across these time points. Luminal epithelial cells and fluid were collected using a cytology brush, and gene expression and metabolite concentrations were assessed by RNAseq and targeted mass spectrometry, respectively. Metabolome profile was similar between treatments within each of days 4, 7, and 14 (false discovery rate (FDR): ≥ 0.1). Concentrations of 53 metabolites changed, independent of treatment, across the diestrus. Metabolites were mostly lipids (40 out 53) and the greatest concentrations were at day 14 (FDR: ≤ 0.1). On day 7, the concentration of putrescine and the gene expression of ODC1, PAOX, SLC3A2, and SAT1 increased (P ≤ 0.05). On day 14, the concentration of 3 ceramides, 4 glucosylceramides, and 12 sphingomyelins and the expression of SGMS2 were increased, in addition to the concentration of choline and 20 phosphatidylcholines. Collectively, the post-estrus concentration of luminal metabolites changed dynamically, independent of the concentration of sex steroids on the previous cycle, and the greatest magnitude changes were on day 14 when lipid metabolism was the most enriched pathway.

Actions of DKK1 on the preimplantation bovine embryo to affect pregnancy establishment, placental function, and postnatal phenotype†.

One mechanism by which the maternal environment regulates the early embryo is by secretion of cell-signaling molecules. One of these is dickkopf WNT signaling pathway inhibitor 1. Objectives were to (A) resolve discrepancies in the literature regarding effects of dickkopf WNT signaling pathway inhibitor 1 in the bovine embryo on development of trophectoderm and competence to establish pregnancy after embryo transfer and (B) determine whether there are long-term consequences of dickkopf WNT signaling pathway inhibitor 1 on placental function and postnatal phenotype. Embryos produced in vitro were cultured with vehicle or 100 ng/mL recombinant human dickkopf WNT signaling pathway inhibitor 1 from Days 5 to 7.5 of development (i.e., the morula and blastocyst stages of development). dickkopf WNT signaling pathway inhibitor 1 increased the number of cells positive for the trophectoderm marker CDX2 at Day 7.5 of development while having no effect on number of cells positive for the inner cell mass marker SOX2. There was no effect of dickkopf WNT signaling pathway inhibitor 1 on pregnancy or calving rate after transfer of blastocysts produced with Y-sorted semen to either lactating dairy cows or suckling beef cows. Treatment with dickkopf WNT signaling pathway inhibitor 1 at the morula-to-blastocyst stages programmed placental function, as measured by an effect of dickkopf WNT signaling pathway inhibitor 1 on plasma concentrations of pregnancy associated glycoproteins and placental lactogen at Day 160 of gestation (although not on other days examined). dickkopf WNT signaling pathway inhibitor 1 treatment also resulted in calves that were heavier at birth as compared to calves derived from control embryos. After birth, dickkopf WNT signaling pathway inhibitor 1 calves grew slower than controls. Results confirm that dickkopf WNT signaling pathway inhibitor 1 alters the developmental program of the bovine embryo to affect both prenatal and postnatal phenotypes.

Cellular responses and microRNA profiling in bovine spermatozoa under heat shock.

In brief: Elevated temperatures disturbed sperm physiology. Bovine sperm cells exposed to heat shock led to diminished mitochondrial activity, fertilizing ability, increased oxidative stress and caspase activity concomitant with a delay in embryonic developmental kinetics and modulation of sperm-borne microRNAsmiRNAs. Abstract: Sperm function is susceptible to adverse environmental conditions. It has been demonstrated that in vivo and in vitro exposure of bovine sperm to elevated temperature reduces sperm motility and fertilizing potential. However, the cascade of functional, cellular, and molecular events triggered by elevated temperature in the mature sperm cell remains not fully understood. Therefore, the aim of this study was to determine the effect of heat shock on mature sperm cells. Frozen-thawed Holstein sperm were evaluated immediately after Percoll purification (0 h non-incubation control) or after incubation at 35, 38.5, and 41°C for 4 h. Heat shock reduced sperm motility after 3-4 h at 41°C while mitochondrial activity was reduced by 38.5 and 41°C when compared to the control. Heat shock also increased sperm reactive oxygen species production and caspase activity. Heat-shocked sperm had lower fertilizing ability, which led to diminished cleavage and blastocyst rates. Preimplantation embryo developmental kinetics was also slowed and reduced by sperm heat shock. The microRNA (miR) profiling identified >300 miRs in bovine sperm. Among these, three and seven miRs were exclusively identified in sperm cells exposed to 35 and 41°C, respectively. Moreover, miR-181d was enriched in sperm cells exposed to higher temperatures. Hence, elevated temperature altered the physiology of mature sperm cells by perturbing cellular processes and the miR profile, which collectively led to lower fertilizing ability and preimplantation development.

Relationship between sperm ubiquitination and equine semen freezability.

This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very low ubiquitin intensity and IV = very high ubiquitin intensity) and location of ubiquitin staining (classification 2). Statistical analyses were performed using SAS software (version 9.4), and p ≤ .05 was considered significant. We observed that PF stallions showed higher percentages (p < .05) of sperm cells with high ubiquitination (11.82% of ubiquitin intensity grade I, 39.13% of ubiquitin intensity grade II, 27.25% of ubiquitin intensity grade III, and 20.67% of grade IV), while GF stallions showed higher percentages (p < .05) of sperm cells with lower staining intensity (28.52% grade I, 59.83% grade II, 7.92% grade III, and 7.02% grade IV). Furthermore, for PF stallions, 23 significant correlations were detected (p < .05) between sperm abnormalities and ubiquitin intensity in different sperm regions. Increased ubiquitination of the sperm head, midpiece, and tail was positively correlated with their respective morphological defects. We concluded that high sperm ubiquitin levels are observed in ejaculates from stallions with poor semen quality (poor freezability), and ubiquitin marking in specific cellular locations can identify sperm morphological defects.

Steroidal Regulation of Oviductal microRNAs Is Associated with microRNA-Processing in Beef Cows.

Information on molecular mechanisms through which sex-steroids regulate oviductal function to support early embryo development is lacking. Here, we hypothesized that the periovulatory endocrine milieu affects the miRNA processing machinery and miRNA expression in bovine oviductal tissues. Growth of the preovulatory follicle was controlled to obtain cows that ovulated a small follicle (SF) and subsequently bore a small corpus luteum (CL; SF-SCL) or a large follicle (LF) and large CL (LF-LCL). These groups differed in the periovulatory plasmatic sex-steroid's concentrations. Ampulla and isthmus samples were collected on day four of the estrous cycle. Abundance of DROSHA, DICER1, and AGO4 transcripts was greater in the ampulla than the isthmus. In the ampulla, transcription of these genes was greater for the SF-SCL group, while the opposite was observed in the isthmus. The expression of the 88 most abundant miRNAs and 14 miRNAs in the ampulla and 34 miRNAs in isthmus were differentially expressed between LF-LCL and SF-SCL groups. Integration of transcriptomic and miRNA data and molecular pathways enrichment showed that important pathways were inhibited in the SF-SCL group due to miRNA control. In conclusion, the endocrine milieu affects the miRNA expression in the bovine oviduct in a region-specific manner.

Heat loss efficiency and HSPs gene expression of Nellore cows in tropical climate conditions.

Adaptation is a relevant characteristic to be understood in livestock animals in order to maintain and raise productivity. In Brazil, the Nellore beef cattle are widely disseminated and well-adapted breed that present good thermoregulatory characteristics for tropical environment conditions. Conversely, the physiological and cellular mechanisms required for thermoregulation and thermotolerance in this breed are still limited. The aim of this study was to comprehend the heat loss efficiency at the whole animal level and heat shock response at the cellular level of Nellore cows in tropical climate conditions. Healthy purebred Nellore cows were classified according to their capacity to lose body heat as Efficient or Inefficient based on vaginal temperature which was continuously monitored by data-loggers. Rectal, tail, and ocular temperatures, sweating rate, and respiratory frequency were collected to assess other thermoregulatory responses. Peripheral mononuclear cells were used for gene expression of heat shock proteins 60, 70, and 90 induced by in vitro heat treatments at 38, 40, and 42 °C. In our findings, the Efficient cows presented higher sweating rates compared to Inefficient cows that presented higher rectal temperature with greater amplitude of vaginal temperature profile. Transcription of the HSP genes was stable at 38 and 40 °C and decreased for all HSP genes at 42 °C. In conclusion, the Nellore efficiency to lose heat was mainly associated with their sweating capacity and cellular thermotolerance confirmed by the maintenance of heat shock proteins transcripts under heat stress. Taken together, this knowledge contributes as a future key for genetic selection of adapted animals.

Sex steroids drive the remodeling of oviductal extracellular matrix in cattle.

The extracellular matrix (ECM) is a group of molecules that offer structural and biochemical support to cells and interact with them to regulate their function. Also, growth factors (GFs) stored in the ECM can be locally released during ECM remodeling. Here, we hypothesize that the balance between ECM components and remodelers is regulated according to the ovarian steroid milieu to which the oviduct is exposed during the periovulatory period. Follicular growth was manipulated to generate cows that ovulated small follicles (SF-small corpus luteum [SCL]; n = 20) or large follicles (LF-large corpus luteum [LCL]; n = 21) and possess corresponding Estradiol (E2) and Progesterone (P4) plasmatic concentrations. Ampulla and isthmus samples were collected on day 4 (day 0 = ovulation induction) and immediately frozen or fixed. The transcriptional profile (n = 3/group) was evaluated by RNA sequencing. MMP Antibody Array was used to quantify ECM remodelers' protein abundance and immunohistochemistry to quantify type I collagen. Transcriptome analysis revealed the over-representation of ECM organization and remodeling pathways in the LF-LCL group. Transcription of ECM components (collagens), remodelers (ADAMs and MMPs), and related GFs were upregulated in LF-LCL. Protein intensities for MMP3, MMP8, MMP9, MMP13, and TIMP4 were greater for the LF-LCL group. Type I collagen content in the mucosa was greater in SF-SCL group. In conclusion, that the earlier and more intense exposure to E2 and P4 during the periovulatory period in LF-LCL animals stimulates ECM remodeling. We speculate that differential ECM regulation may contribute to oviductal receptivity to the embryo.

Sex steroids modulate morphological and functional features of the bovine oviduct.

In cattle, the oviduct plays a major role in the reproductive process; however, molecular control of oviduct receptivity to the embryo is poorly understood. A model for receptivity based on size of the pre-ovulatory follicle (POF) was used to compare oviductal morphology, cellular proliferation, and candidate transcript abundance. Growth of the POF of Nelore (Bos indicus) cows was manipulated to produce two groups: a large POF-large corpus luteum (CL) group (LF-LCL; greater receptivity) and a small POF-small CL group (SF-SCL). Samples of the ampulla and isthmus ipsilateral and contralateral to CL were collected 4 days after GnRH-induced ovulation. Tissues were either embedded in paraffin for Harris-Hematoxylin and Eosin and periodic acid-Schiff staining and KI67 immunostaining, followed by morphological analyses, or stored at -80 °C for RNA extraction, cDNA synthesis, and qPCR analyses. The effects of group (LF-LCL and SF-SCL), region (ampulla and isthmus), and side (ipsilateral and contralateral) were analyzed using three-way nested ANOVA. The ipsilateral ampulla of the LF-LCL group presented more primary mucosal folds, a greater mucosal-folding grade and luminal perimeter, and more secretory cells and proliferating cells when compared with the ampulla of the SF-SCL group and with the contralateral ampulla of both groups. There were no morphological differences in the isthmus between groups and sides. Changes in transcript abundance are suggestive of LF-LCL-stimulated secretory activity. In summary, ovulation of a larger POF generates a periovulatory endocrine milieu that modulates morphological and functional features of the bovine oviduct which may support embryo survival and development.

Regulation of the polyamine metabolic pathway in the endometrium of cows during early diestrus.

The timing and magnitude of exposure to preovulatory estradiol followed by post-ovulatory progesterone (periovulatory endocrine milieu) in cattle modulate endometrial gene expression, histotroph composition, and conceptus development, but the mechanisms underlying this regulation remain unknown. Using an experimental model based on the modulation of follicle growth, this work aimed to evaluate if the polyamine metabolic pathway is regulated by the periovulatory endocrine milieu. Nelore cows were manipulated to ovulate small (n = 15) or large (n = 15) follicles, then the profiles of polyamines and their synthetic enzymes were compared between groups. Transcripts for the enzymes of this pathway, ornithine decarboxylase 1 (ODC1; the rate-limiting enzyme in polyamine biosynthesis) protein quantification, adenosylmethionine decarboxylase 1 (AMD1) protein immunolocalization, and concentrations of the different polyamines (putrescine, spermidine, and spermine) were respectively quantified by quantitative reverse-transcriptase PCR, immunoblotting, immunohistochemistry, and gas chromatography-mass spectrometry in both the endometrium and uterine flushing. No differences in gene and protein expression or concentration of polyamines were observed between groups. There were significant correlations between the relative abundance of ODC1 and spermidine/spermine N1-acetyltransferase 1 (SAT1) transcripts as well as between antizyme inhibitor 1 (AZIN1) and adenosylmethionine decarboxylase 1 (AMD1) transcripts. In conclusion, our results show that the polyamine metabolic pathway is present and functional, but not regulated by the periovulatory endocrine milieu in the bovine endometrium.

Seasonal environmental fluctuations alter the transcriptome dynamics of oocytes and granulosa cells in beef cows.

BACKGROUND: Examining the mechanistic cellular responses to heat stress could aid in addressing the increasing prevalence of decreased fertility due to elevated ambient temperatures. Here, we aimed to study the differential responses of oocytes and granulosa cells to thermal fluctuations due to seasonal differences. Dry beef cows (n = 10) were housed together, synchronized and subjected to a stimulation protocol to induce follicular growth before ovum pick-up (OPU). Two OPU's were conducted (summer and winter) to collect cumulus-oocyte-complexes (COCs) and granulosa cells. In addition, rectal temperatures and circulating blood samples were collected during OPU. Oocytes were separated from the adherent cumulus cells, and granulosa cells were isolated from the collected OPU fluid. RNA was extracted from pools of oocytes and granulosa cells, followed by library preparation and RNA-sequencing. Blood samples were further processed for the isolation of plasma and leukocytes. The transcript abundance of HSP70 and HSP90 in leukocytes was evaluated using RT-qPCR, and plasma cortisol levels were evaluated by immunoassay. Environmental data were collected daily for three weeks before each OPU session. Data were analyzed using MIXED, Glimmix or GENMOD procedures of SAS, according to each variable distribution. RESULTS: Air temperatures (27.5 °C vs. 11.5 °C), average max air temperatures (33.7 °C vs. 16.9 °C), and temperature-humidity indexes, THI (79.16 vs. 53.39) were shown to contrast significantly comparing both the summer and winter seasons, respectively. Rectal temperatures (Summer: 39.2 ± 0.2 °C; Winter: 38.8 ± 0.2 °C) and leukocyte HSP70 transcript abundance (Summer: 4.18 ± 0.47 arbitrary units; Winter: 2.69 ± 0.66 arbitrary units) were shown to increase in the summer compared to the winter. No visual differences persisted in HSP90 transcript abundance in leukocytes and plasma cortisol concentrations during seasonal changes. Additionally, during the summer, 446 and 940 transcripts were up and downregulated in oocytes, while 1083 and 1126 transcripts were up and downregulated in the corresponding granulosa cells, respectively (Fold Change ≤ -2 or ≥ 2 and FDR ≤ 0.05). Downregulated transcripts in the oocytes were found to be involved in ECM-receptor interaction and focal adhesion pathways, while the upregulated transcripts were involved in protein digestion and absorption, ABC transporters, and oocyte meiosis pathways. Downregulated transcripts in the granulosa cells were shown to be involved in cell adhesion molecules, chemokine signaling, and cytokine-cytokine receptor interaction pathways, while those upregulated transcripts were involved in protein processing and metabolic pathways. CONCLUSION: In conclusion, seasonal changes dramatically alter the gene expression profiles of oocytes and granulosa cells in beef cows, which may in part explain the seasonal discrepancies in pregnancy success rates during diverging climatic weather conditions.

Embryo production by Holstein heifers superovulated with a recombinant long-acting follicle stimulating hormone analog.

This study aimed to evaluate embryo yield in Holstein heifers superovulated with a single injection of recombinant, long-acting human follicle-stimulating hormone (rFSH) versus multiple injections of pituitary-derived follicle-stimulating hormone (FSH). In Experiment 1, heifers were assigned randomly to one of 4 experimental groups: Control (280 mg of pituitary derived FSH; six injections of 40 mg and two injections of 20 mg, each ~12 h apart, n = 16); rFSH1 (50 µg of FSH analog protein, n = 16); rFSH2 (75 µg of FSH analog protein, n = 16) or; rFSH3 (100 µg of FSH analog protein, n = 16). The rFSH was administered as a single injection. Estrous cycles were presynchronized with gonadorelin acetate (GnRH) and an intra-vaginal progesterone insert (CIDR) on d -8, followed by cloprostenol sodium (PGF2α) on d -3 and d -2 with removal of the CIDR, and GnRH on d 0. On d 5, ovarian follicles ≥ 5 mm in diameter were ablated by transvaginal ultrasound-guided aspiration. On d 6.5, heifers received a CIDR and the rFSH or the first injection of pituitary-derived FSH. On d 9, heifers received two injections of PGF2α, 12 h apart. On d 10.5, the CIDR was removed, and on d 11, heifers received a GnRH injection. Heifers were AI-inseminated 12- and 24-hours post-GnRH injection, and uterine contents were flushed trans-cervically on day 18 (7 d after the GnRH injection). Ovarian follicles ≥ 5 mm and corpora lutea were counted via ultrasound on days 5, 9, and 18. In Experiment 1, group did not affect (P = 0.52) the number of follicles ≥ 5 mm (Control = 15.9 ± 1.2; rFSH1 = 17.5 ± 1.3; rFSH2 = 17.1 ± 1.3; rFHS 3 = 18.6 ± 1.4 follicles) or the number of corpora lutea (P = 0.96) on d 9 (Control = 1.1 ± 0.3; rFSH1 = 1.1 ± 0.3; rFSH2 = 1.1 ± 0.3; rFSH3 = 0.9 ± 0.2). Furthermore, there was no effect (P = 0.28) of rFSH dose on freezable embryos (grade 1 and 2 embryos) collected on d 18 (Control = 4.7 ± 1.1; rFSH1 = 4.7 ± 1.2; rFSH2 = 4.4 ± 1.1; rFSH3 = 2.6 ± 0.7 embryos). In Experiment 2, Control (n=8) and rFSH1 (n=16) groups were repeated in 3 replicates using the same protocols as Experiment 1. Consequently, Results showed that rFSH produced fewer total number of ova/embryos (Control = 9.9 ± 1.5 vs. rFHS1 = 5.9 ± 0.9, P = 0.04) and fewer freezable embryos (Control = 5.3 ± 1.0 vs. rFSH1 = 1.4 ± 0.3, P < 0.01). In conclusion, the single rFSH injection effectively induced superovulation; however, its repeated use reduced embryo production.

Impact of Supplementing a Backgrounding Diet with Nonprotein Nitrogen on In Vitro Methane Production, Nutrient Digestibility, and Steer Performance.

Two experiments were conducted to evaluate the effect of nonprotein nitrogen (NPN) supplementation on in vitro fermentation and animal performance using a backgrounding diet. In experiment 1, incubations were conducted on three separate days (replicates). Treatments were control (CTL, without NPN), urea (U), urea-biuret (UB), and urea-biuret-nitrate (UBN) mixtures. Except for control, treatments were isonitrogenous using 1% U inclusion as a reference. Ruminal fluid was collected from two Angus-crossbred steers fed a backgrounding diet plus 100 g of a UBN mixture for at least 35 d. The concentration of volatile fatty acids (VFA) and ammonia nitrogen (NH3-N), in vitro organic matter digestibility (IVOMD), and total gas and methane (CH4) production were determined at 24 h of incubation. In experiment 2, 72 Angus-crossbred yearling steers (303 ±â€…29 kg of body weight [BW]) were stratified by BW and randomly allocated in nine pens (eight animals/pen and three pens/treatment). Steers consumed a backgrounding diet formulated to match the diet used in the in vitro fermentation experiment. Treatments were U, UB, and UBN and were isonitrogenous using 1% U inclusion as a reference. Steers were adapted to the NPN supplementation for 17 d. Then, digestibility evaluation was performed after 13 d of full NPN supplementation for 4 d using 36 steers (12 steers/treatment). After that, steer performance was evaluated for 56 d (24 steers/treatment). In experiment 1, NPN supplementation increased the concentration of NH3-N and VFA (P < 0.01) without affecting the IVOMD (P = 0.48), total gas (P = 0.51), and CH4 production (P = 0.57). Additionally, in vitro fermentation parameters did not differ (P > 0.05) among NPN sources. In experiment 2, NPN supplementation did not change dry matter and nutrient intake (P > 0.05). However, UB and UBN showed lower (P < 0.05) nutrient digestibility than U, except for starch (P = 0.20). Dry matter intake (P = 0.28), average daily gain (P = 0.88), and gain:feed (P = 0.63) did not differ among steers receiving NPN mixtures. In conclusion, tested NPN mixtures have the potential to be included in the backgrounding diets without any apparent negative effects on animal performance and warrant further studies to evaluate other variables to fully assess the response of feeding these novel NPN mixtures.

Nonprotein nitrogen (NPN) supplements can be used as a nitrogen source for ruminants fed low-protein diets. The most common NPN source is urea, included typically at a range between 0.5% and 1% of the diet dry matter in growing beef cattle. Although other NPN sources and mixtures are available, there is scarce information regarding their use in ruminant production. Two experiments were conducted to evaluate the effect of NPN sources on in vitro fermentation and animal performance using a backgrounding diet. In experiment 1, three different incubations were performed for 24 h. Treatments were control (without NPN), urea (U), urea­biuret (UB), and urea­biuret­nitrate (UBN) mixtures. In experiment 2, 72 crossbred yearling steers were randomly assigned to one of the following treatments: U, UB, and UBN mixtures. Diets were formulated to contain the same nitrogen concentration in both experiments. In experiment 1, supplementation of NPN increased the in vitro fermentation, but there were no differences among NPN sources. In experiment 2, steers performed similarly among NPN sources. These findings suggest that NPN mixtures have the potential to be included in the backgrounding diets without detrimental effects. Further studies should evaluate other variables (e.g., fermentation dynamic and microbial protein supply) when using these novel mixtures.

Artificial shade as a heat abatement strategy to grazing beef cow-calf pairs in a subtropical climate.

Grazing livestock in subtropical and tropical regions are susceptible to prolonged exposition to periods of extreme environmental conditions (i.e., temperature and humidity) that can trigger heat stress (HS). Currently, there is limited information on the effects of HS in the cow-calf sector globally, including in the southern U.S., as well as on mitigation strategies that could be implemented to improve animal well-being and performance. This study evaluated the impact of artificial shade (SHADE vs. NO SHADE) and breed (ANGUS vs. BRANGUS) on performance of pregnant-lactating cows, nursing heifers, and their subsequent offspring. Twenty-four Angus and 24 Brangus black-hided cows [579 ± 8 kg body weight (BW); approximately 85 d of gestation] and their nursing heifers (approximately 174 d of age) were randomly allocated to 12 'Pensacola' bahiagrass pastures (Paspalum notatum Flüggé; 1.3 ha, n = 4 pairs/pasture), with or without access to artificial shade [NO SHADE BRANGUS (NSB), NO SHADE ANGUS (NSA), SHADE BRANGUS (SB), and SHADE ANGUS (SA)] for 56 d that anticipated weaning during the summer season in Florida. Body condition score (BCS) of cows, blood samples, and BW of cow-calf pairs were obtained every 14 d during the 56-d experimental period until weaning. Following weaning (d 56), treatments were ceased, and cows and weaned heifers were managed alike. Weaned heifers were randomly allocated to 4 pens (n = 12/pen) equipped with GrowSafe feed bunks for 14 d to assess stress responses during weaning via plasma haptoglobin. An effect of SHADE × BREED interaction was detected for cow ADG, BW change, final BW, and final BCS, where SB had the greatest ADG, BW change, final BW, and final BCS. On d 14, SA cows had the greatest concentrations of insulin whereas on d 28 NSB had the lowest concentrations, NSA the greatest, and SA and SB being intermediate. On d 56, SA tended to have the greatest plasma insulin concentrations and SB the lowest. Weight gain per area (kg/ha) tended to be 11.4 kg/ha greater in SHADE vs. NO SHADE pastures. Pre-weaning calf ADG tended to be 0.14 kg greater for SHADE vs. NO SHADE calves. Weaning weight and BW at 14-d post-weaning were lesser for NSB vs. NSA, SA, and SB, whereas no differences in postweaning ADG or haptoglobin were observed. Effects of SHADE × BREED × day interaction was detected on plasma concentrations of IGF-1, in which NSA heifers had the lowest concentrations on weaning day. Gestation length was greater for SHADE vs. NO SHADE cows, but with no impacts on subsequent calf birth and weaning weight. In summary, providing artificial shade to pregnant-lactating beef cows increased body weight gain of nursing heifers and Brangus cows, while no impact on Angus dams were observed. The provision of artificial shade during the first trimester of gestation did not alter growth performance of the subsequent offspring at birth and weaning even though gestation length was longer.

Extracellular vesicle-microRNAs mediated response of bovine ovaries to seasonal environmental changes.

BACKGROUND: Among the various seasonal environmental changes, elevated ambient temperature during the summer season is a main cause of stress in dairy and beef cows, leading to impaired reproductive function and fertility. Follicular fluid extracellular vesicles (FF-EVs) play an important role in intrafollicular cellular communication by, in part, mediating the deleterious effects of heat stress (HS). Here we aimed to investigate the changes in FF-EV miRNA cargoes in beef cows in response to seasonal changes: summer (SUM) compared to the winter (WIN) season using high throughput sequencing of FF-EV-coupled miRNAs. In addition to their biological relevance, the potential mechanisms involved in the packaging and release of those miRNAs as a response to environmental HS were elucidated. RESULTS: Sequencing analysis revealed that an average of 6.6% of the EV-RNA mapped reads were annotated to bovine miRNAs. Interestingly, miR-148a, miR-99a-5p, miR-10b, and miR-143 were the top four miRNAs in both groups accounting for approximately 52 and 62% of the total miRNA sequence reads in the SUM and WIN groups, respectively. A group of 16 miRNAs was up-regulated and 8 miRNAs were down-regulated in the SUM compared to the WIN group. Five DE-miRNAs (miR-10a, miR-10b, miR-26a, let-7f, and miR-1246) were among the top 20 expressed miRNA lists. Sequence motif analysis revealed the appearance of two specific motifs in 13 out of the 16 upregulated miRNAs under HS conditions. Both motifs were found to be potentially bonded by specific RNA binding proteins including Y-box binding proteins (YBX1 and YBX2) and RBM42. CONCLUSION: Our findings indicate that FF EV-coupled miRNA profile varies under seasonal changes. These miRNAs could be a good indicator of the cellular mechanism in mediating HS response and the potential interplay between miRNA motifs and RNA binding proteins can be one of the mechanisms governing the packaging and release of miRNAs via EVs to facilitate cellular survival.

Evaluation of carinata meal or cottonseed meal as protein sources in silage-based diets on behavior, nutrient digestibility, and performance in backgrounding beef heifers.

Changing climatic conditions are imposing risks and diminishing yields in agriculture. Sorghum (Sorghum bicolor) silage is a feasible option for backgrounding beef cattle in terms of economic risk management and animal productivity when compared with corn (Zea mays) silage, due to its drought adaptability. Similarly, Brassica carinata meal has proven to be a viable alternative as a protein supplement in forage-based beef cattle systems, when included at 10% of the diet dry matter (DM). However, research is scarce regarding its inclusion in silage-based diets for backgrounding animals. The objective of this trial was to compare a processor-chopped sorghum silage (SS) against a typical corn silage (CS) in a digestibility and performance trial while supplementing two protein sources; one traditionally used like cottonseed meal (CSM) and one novel like B. carinata meal (BCM). A total of 84 Angus crossbred heifers (307 ± 33 kg BW) were evaluated in a randomized block design with a 2 × 2 factorial treatment arrangement with type of silage and protein source as factors. Diets were fed ad libitum, consisting of 89% silage source plus 10% protein source, and 1% mineral inclusion on DM basis. The experimental period consisted of 14 d of adaptation followed by 5 d of apparent total tract digestibility measurements and 56 d of animal performance and intake behavior measurements. Heifers fed SS showed greater number of daily meals but decreased meal sizes (P ≤ 0.05), not differing in meal length (P > 0.10) when compared with CS. Dry matter and organic matter (OM) digestibility showed a silage type × protein source interaction (P ≤ 0.01), where in CS diets, OM tended to be more digestible with CSM vs. BCM, and it did not differ between protein sources in SS based diets. There was an effect of protein (P ≤ 0.01) on ADF digestibility, where CSM was greater than BCM. No effect of treatment was observed (P ≥ 0.10) on DM intake. Average daily gain (ADG) and gain-to-feed ratio were greater for CS than SS (P ≤ 0.01) regardless of protein source. Although heifers fed CS had greater feed efficiency and digestibility, SS can still be considered a viable option for backgrounding beef heifers, obtaining adequate ADG rates of 0.945 kg/d. Lastly, BCM did not differ from CSM in terms of feed efficiency and animal performance, proving to be a viable alternative protein source in silage-based diets.

Increased atmospheric CO2, rising temperatures, and altered patterns of precipitation can limit the production of certain crops commonly used in agriculture, increasing risk, cost, and availability of feedstuffs. The search for alternative plants that could thrive in these changing scenarios is necessary to provide producers with a broader array of options to feed cattle. In this study, sorghum silage was compared with corn silage as the main dietary ingredient, with either Brassica carinata (carinata) or cottonseed meal as the protein source for growing beef heifers. Variables assessed included intake behavior, digestibility, and performance of beef heifers. Heifers fed sorghum silage gained less than heifers fed corn silage, though they grew at an adequate rate for a replacement heifer. Carinata meal showed similar performance results compared with cottonseed meal, despite some of its fiber components being less digestible in the total tract. Therefore, sorghum silage has potential to be a viable feedstuff for growing beef heifers although it may result in decreased performance compared with corn silage. Alternatively, carinata meal can be a practical alternative to cottonseed meal as a protein source in terms of animal performance. This could translate in an increase in the planted area of both sorghum and carinata in Southern United States, as they are adapted to drought and high temperatures, enhancing the resilience of beef production systems in a context of increased climate variability.