Transcriptome changes associated with elongation of bovine conceptuses I: Differentially expressed transcripts in the conceptus on day 17 after insemination.

The objective was to characterize transcriptome changes associated with elongation in bovine conceptuses during preimplantation stages. Nonlactating Holstein cows were euthanized 17 d after artificial insemination (AI) and the uterine horn ipsilateral to the CL was flushed with saline solution. Recovered conceptuses were classified as small (1.2 to 6.9 cm; n = 9), medium (10.5 to 16.0 cm; n = 9), or large (18.0 to 26.4 cm; n = 10). Total mRNA was extracted and subjected to transcriptome analyses using the Affymetrix Gene Chip Bovine array. Data were normalized using the GCRMA method and analyzed by robust regression using the Linear Models for Microarray library within Bioconductor in R. Transcripts with P ≤ 0.05 after adjustment for false discovery rate and fold change ≥1.5 were considered differentially expressed. Functional analyses were conducted using the Ingenuity Pathway Analysis platform. Comparisons between large versus small (LvsS), large versus medium (LvsM), and medium versus small (MvsS) conceptuses yielded a total of 634, 240, and 63 differentially expressed transcripts, respectively. Top canonical pathways of known involvement with embryo growth that were upregulated in large conceptuses included actin cytoskeleton (LvsS), integrin signaling (LvsS and LvsM), ephrin receptor (LvsS), mesenchymal transition by growth factor (LvsM), and regulation of calpain protease (LvsS). Transcripts involved with lipid metabolism pathways (LXR/RXR, FXR/RXR, hepatic fibrosis) were associated with the LvsS and LvsM, and some transcripts such as APOC2, APOH, APOM, RARA, RBP4, and PPARGC1A, were involved in these pathways. An overall network summary associated biological downstream effects of invasion of cells, proliferation of embryonic cells, and inhibition of organismal death in the LvsS. In conclusion, differently expressed transcripts in the LvsS comparison were associated with the cell growth, adhesion, and organismal development, although part of these findings could be attributed to differences in circulatory concentrations of progesterone of the cows that bore large and small conceptuses. The large and medium conceptuses developed under similar concentrations of progesterone and presented 240 differently expressed transcripts, associated with cell differentiation, metabolite regulation, and other biological processes.

Transcriptome changes associated with elongation of bovine conceptuses II: Differentially expressed transcripts in the endometrium on day 17 after insemination.

The objective was to characterize endometrial transcriptome on d 17 of gestation in dairy cows according to conceptus length. Nonlactating Holstein cows (n = 48) were slaughtered 17 d after AI and the uterine horn ipsilateral to the corpus luteum (CL) was flushed with saline solution. Recovered conceptuses were classified as small (1.2-6.9 cm; n = 9), medium (10.5-16.0 cm; n = 9), or large (18.0-26.4 cm; n = 10). Samples of intercaruncular endometrium dissected from the caudal, intermediate, and cranial portions of the uterine horn ipsilateral to the pregnancy were pooled for analyses. Total mRNA was extracted from endometrial tissue and subjected to transcriptome analyses using the Affymetrix Gene Chip Bovine array. Data were normalized using the GCRMA method and analyzed by robust regression using the Linear Models for Microarray library within Bioconductor in R. Transcripts with P ≤ 0.05 after adjustment for false discovery rate and fold change ≥1.5 were considered differentially expressed. Functional analyses were conducted using the Ingenuity Pathway Analysis platform. Comparisons between endometria of cows carrying large versus small (LvsS), large versus medium (LvsM), and medium versus small (MvsS) conceptuses yielded a total of 235, 21, and 94 differentially expressed transcripts, respectively. Top canonical pathways included the antigen presentation pathway and Th1/Th2 activation pathways, both for LvsS and MvsS. Interferon-α and -γ were identified as activated upstream regulators, primarily based on differently expressed transcripts such as IDO1, ISG20, WARS, LGALS9, IFI44, and PSMB9 (LvsS and MvsS). For LvsS, regulator analyses revealed predicted activation of FOXO1, IFN, NFACTC2, IL-12, IL-6, and IL-18, whereas it depicted inhibition of IL10RA and ZBTB1. Changes in these regulators were associated with a downstream activation of leukocytes, as well as quantity and expansion of T lymphocytes. Canonical pathways associated with the comparison LvsM included cell cycle G2/M DNA damage checkpoint regulation, cell cycle control of chromosomal replication. Moreover, tretinoin was predicted, as activated in upstream analysis for the same comparison. In conclusion, most of the differently expressed transcripts in the endometrium on d 17 of gestation were identified between cows carrying small conceptuses compared with counterparts carrying medium and large conceptuses and were involved with pathways associated with modulation of the immune response.

A randomized controlled trial examining the effects of treatment with propylene glycol and injectable cyanocobalamin on naturally occurring disease, milk production, and reproductive outcomes of dairy cows diagnosed with concurrent hyperketonemia and hypoglycemia.

The objective of this study was to assess the effects of treatment with propylene glycol (PG) and cyanocobalamin (B12) on health, milk production, and reproductive outcomes of cows diagnosed with hyperketonemia (HK), hypoglycemia (HG), or concurrent HKHG. Glucose and ß-hydroxybutyric acid (BHBA) concentrations were assessed in whole blood using a handheld device in lactating dairy cows (n = 2,418) between 3 and 9 d postpartum. Cows categorized as HK (n = 232, BHBA ≥1.2 mmol/L), HG (n = 161, glucose ≤2.2 mmol/L), and concurrent HKHG (n = 204, BHBA ≥1.2 mmol/L, and glucose ≤2.2 mmol/L) were randomized to receive treatment or to remain untreated (control). Treatment consisted of a single dose of B12 (10 mg, intramuscularly) and 300 mL of PG orally for 5 d, starting on the day of cow-side testing. Milk production, health, and reproductive outcomes were analyzed according to groups. Statistical analysis was carried out using SAS version 9.4 (SAS/STAT, SAS Institute Inc.). Treatment in HG cows decreased clinical ketosis, increased milk production in the fifth week of lactation for multiparous cows, and tended to increase 305-d mature-equivalent milk yield (305ME) for primiparous cows compared with untreated cows with the same metabolic profile. For cows with HKHG, treatment increased 305ME in multiparous cows and tended to increase 305ME in primiparous cows. No differences were found for treatment among any of the metabolic groups regarding reproductive outcomes, nor were any treatment effects found among HK cows. Glycemic status may help identify metabolically challenged early postpartum dairy cows, which may have differential response to PG and B12 treatment.

Characterization of metabolic profile, health, milk production, and reproductive outcomes of dairy cows diagnosed with concurrent hyperketonemia and hypoglycemia.

The objective of this prospective cohort study was to characterize the metabolic profile, health, milk production, and reproductive outcomes of cows diagnosed with hyperketonemia (HK; ß-hydroxybutyrate ≥1.2 mmol/L), hypoglycemia (HG; glucose ≤2.2 mmol/L), or concurrent HK and HG (HKHG). Glucose and ß-hydroxybutyrate concentrations in whole blood were assessed using a handheld device (Precision Xtra, Abbott Laboratories) in lactating dairy cows (n = 2,418) between 3 and 9 d postpartum. Cows were categorized into 4 groups: no HK or HG (healthy; Norm = 1,821), HK only (HK = 232), HG only (HG = 161), and concurrent HK and HG (HKHG = 204). Subsequent milk production, along with health and reproductive outcomes, as recorded by farm personnel, were analyzed according to metabolic category. Serum collected on the day of cow-side diagnosis of hyperketonemia and hypoglycemia was evaluated for total calcium (tCa), magnesium (Mg), nonesterified fatty acids (NEFA), triglycerides (TG), and urea using an automated chemistry analyzer (Randox Daytona; Randox Laboratories Ltd.). Statistical analysis was carried out using SAS version 9.4 (SAS Institute Inc.). Hyperketonemia in multiparous cows was associated with greater incidence of metabolic abnormalities (hypomagnesemia, hypocalcemia, and elevated NEFA and urea). Hyperketonemia in primiparous and multiparous cows led to increased adverse health events (culling rate, retained fetal membranes, puerperal metritis, clinical ketosis, left displaced abomasum) relative to Norm cows. Multiparous cows with HKHG had fewer metabolic disturbances (hypomagnesemia, hypocalcemia, elevated NEFA) than HK cows. Cows with HKHG had an increased incidence of clinical ketosis and left displaced abomasum relative to Norm cows. Cows with HG had similar metabolic profiles to Norm cows and had lower incidence of retained fetal membranes and puerperal metritis than cows with HK. Multiparous cows with HG produced more milk than Norm cows from wk 10 to 20, whereas multiparous cows with HK produced less milk than Norm cows. For primiparous cows, HK did not have a negative effect on milk production compared with Norm cows.

Effect of nerve growth factor-ß administered at insemination for lactating Holstein dairy cows bred after timed-artificial insemination protocol.

The objectives of this study were to determine the effects of nerve growth factor-ß (NGF), purified from bulls' seminal plasma and administered at the time of artificial insemination (AI), on progesterone post-AI, interferon-stimulated genes (ISG), and pregnancy per AI (P/AI) for lactating Holstein dairy cows enrolled in a timed-AI protocol. We hypothesized that administration of NGF at the time of AI would increase plasma progesterone post-AI, upregulate relative abundance of ISG, and improve P/AI in lactating dairy cows. Holstein cows (n = 557) from a single commercial dairy farm were blocked by parity and randomly assigned to receive an intramuscular injection containing 296 µg of bovine purified NGF at the time of AI, diluted in 2 mL of phosphate-buffered saline (NGF: n = 275), or receive only the 2 mL of phosphate-buffered saline (control: n = 282). Plasma progesterone and corpus luteum size were assessed in a subset of cows (NGF: n = 32; control: n = 36) at d 7, 14, and 19 post-AI. Relative mRNA abundance of ISG (ISG15, MX1, MX2, and RTP4) was assessed in peripheral blood leukocytes on d 19 post-AI. Pregnancy diagnosis was performed at 37 and 65 d post-AI. There was an interaction effect between treatment and parity for plasma progesterone; however, plasma progesterone and ISG did not differ between treatments. There were no effects of NGF for P/AI at 37 d post-AI (NGF = 40.0% vs. control = 41.6%), 65 d post-AI (NGF = 36.0% vs. control = 38.1%), and for pregnancy loss (NGF = 8.4% vs. control = 7.7%). The current study revealed that effects to NGF in lactating Holstein cows were minor and contingent with parity for progesterone, and no improvement in ISG relative abundance and P/AI were observed.

Effect of gonadotropin-releasing hormone administered at the time of artificial insemination for cows detected in estrus by conventional estrus detection or an automated activity-monitoring system.

The objectives of this study were to determine the effects of GnRH at the time of artificial insemination (AI) on ovulation, progesterone 7 d post-AI, and pregnancy in cows detected in estrus using traditional methods (tail chalk removal and mount acceptance visualization) or an automated activity-monitoring (AAM) system. We hypothesized that administration of GnRH at the time of AI would increase ovulation rate, plasma progesterone post-AI, and pregnancy per AI (P/AI) in cows detected in estrus. In experiment 1, Holstein cows (n = 398) were blocked by parity and randomly assigned to receive an injection of GnRH at the time of estrus detection/AI (GnRH, n = 197) or to remain untreated (control, n = 201) on 4 farms. The GnRH was administered as 100 µg of gonadorelin acetate. Ovarian structures and plasma progesterone were assessed in a subset of cows (GnRH, n = 52; control, n = 55) in experiment 1 at the time of AI and 7 d later. In experiment 2, a group of 409 cows in an AAM farm were enrolled as described for experiment 1 (GnRH, n = 207; control, n = 202). Data were categorized for parity (primiparous vs. multiparous), season (cool vs. warm), number of services (first vs. > first), DIM (>150 DIM vs. ≤150 DIM), and for AAM cows in experiment 2 for activity level (high: 90-100 index vs. low: 35-89 index). Pregnancy diagnosis was performed between 32 and 45 d post-AI (P1) and 60 to 115 d post-AI (P2). In experiment 1, there was no difference in plasma progesterone at day of estrus detection (control = 0.09 ng/mL vs. GnRH = 0.16 ng/mL), 7 d later (control = 2.03 ng/mL vs. GnRH = 2.18 ng/mL), and ovulation rate (GnRH = 83.2% vs. control = 77.9%) between treatments. There were no effects of GnRH in experiment 1 for P/AI at P1 (control = 43.3% vs. GnRH = 38.6%), P2 (control = 38.4% vs. GnRH = 34.5%), and for pregnancy loss (control = 9.8% vs. GnRH = 8.2%). In experiment 2, there were no effects of GnRH for P/AI at P1 (control = 39.6% vs. GnRH = 40.1%), P2 (control = 35.0% vs. GnRH = 37.4%), and for pregnancy loss (control = 9.5% vs. GnRH = 6.2%). There was a tendency for a parity effect on P/AI for P1, but not P2 or for pregnancy loss. High-activity cows had greater P/AI in P1 (low activity = 27.9% vs. high activity = 44.1%), P2 (low activity = 21.8% vs. high activity = 41.2%), and lower pregnancy loss (low activity = 20.7% vs. high activity = 5.1%), but there were no interactions between treatment and activity level. The current study did not support the use of GnRH at estrus detection to improve ovulatory response, progesterone 1 wk post-AI, and P/AI. More research is needed to investigate the relationship between GnRH at the time of AI and activity level in herds using AAM systems.

Characterization of pregnancy-associated glycoproteins and progesterone as a predictor of twins and conceptus loss in high-risk-pregnancy Holstein cows.

The objective of this study was to characterize plasma concentrations of pregnancy-associated glycoprotein (PAG) and progesterone (P4) as predictors of twins and pregnancy loss in Holstein cows with high-risk pregnancies. High-risk pregnancy was characterized using transrectal ultrasonography 37 d after artificial insemination (AI) based on the following criteria: small embryo size <15 mm, n = 10), slow heartbeat (<60 beats per minute, n = 11), and extra-amniotic membrane (additional amniotic membrane, n = 3). A cohort of twins (n = 41) diagnosed at d 37 post-AI was also enrolled. Each high-risk and twin pregnancy cow was paired with a cow of the same parity carrying a normal singleton at d 37 post-AI (control, n = 65). Blood samples were collected to measure PAG and P4 at d 37, 44, and 51 post-AI. Statistical analysis was performed using ANOVA, logistic regression, and receiver operator characteristic (ROC) curve with JMP software (SAS Institute Inc., Cary, NC). Pregnancy loss at d 51 post-AI was greater in high-risk pregnancies than in controls and twins (control = 1.5%; high-risk = 87.5%; twins = 12.2%). Concentration of PAG at d 37 post-AI did not differ among groups (control = 5.3 ± 0.7; high-risk = 4.8 ± 1.2; twins = 4.0 ± 0.9 ng/mL). Concentration of P4 at d 37 post-AI was greater in twins than in high-risk pregnancies and control, and lesser in high-risk pregnancies than in controls (control = 7.0 ± 0.3; high-risk = 5.9 ± 0.4; twins = 8.4 ± 0.3 ng/mL). Regression and ROC analysis for PAG at d 37 post-AI did not find a threshold to predict pregnancy loss or twins. Regression and ROC analysis for P4 at d 37 post-AI found that a threshold of 6.5 ng/mL predicted pregnancy loss with an area under the curve (AUC) of 0.64, and a threshold of 7.2 ng/mL predicted twins with AUC of 0.71. In summary, pregnancy loss and twins were predicted with only moderate accuracy by P4 concentration at d 37 post-AI, and the variability in PAG concentrations at d 37 post-AI was not sufficient to generate a threshold to predict pregnancy loss and twins in Holstein lactating cows.

A myosin-Va tail fragment sequesters dynein light chains leading to apoptosis in melanoma cells.

Previous studies proposed that myosin-Va regulates apoptosis by sequestering pro-apoptotic Bmf to the actin cytoskeleton through dynein light chain-2 (DLC2). Adhesion loss or other cytoskeletal perturbations would unleash Bmf, allowing it to bind and inhibit pro-survival Bcl2 proteins. Here, we demonstrated that overexpression of a myosin-Va medial tail fragment (MVaf) harboring the binding site for DLC2 dramatically decreased melanoma cell viability. Morphological and molecular changes, including surface blebbing, mitochondrial outer membrane permeabilization, cytochrome-c and Smac release, as well as caspase-9/-3 activation and DNA fragmentation indicated that melanoma cells died of apoptosis. Immobilized MVaf interacted directly with DLCs, but complexed MVaf/DLCs did not interact with Bmf. Overexpression of DLC2 attenuated MVaf-induced apoptosis. Thus, we suggest that, MVaf induces apoptosis by sequestering DLC2 and DLC1, thereby unleashing the pair of sensitizer and activator BH3-only proteins Bmf and Bim. Murine embryonic fibroblasts (MEFs) lacking Bim and Bmf or Bax and Bak were less sensitive to apoptosis caused by MVaf expression than wild-type MEFs, strengthening the putative role of the intrinsic apoptotic pathway in this response. Finally, MVaf expression attenuated B16-F10 solid tumor growth in mice, suggesting that this peptide may be useful as an apoptosis-inducing tool for basic and translational studies.