Nobiletin-induced partial abrogation of deleterious effects of AKT inhibition on preimplantation bovine embryo development in vitro†.

During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2- to 8-cell stage, MNEGA) or major (8- to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15 µM AKT-InhIII; 10 µM AKT-InhIV; 10 µM nobiletin; nobiletin + AKT-InhIII; nobiletin + AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors, which infers that nobiletin probably uses another signaling cascade that PI3K/AKT during early embryo development in bovine.

Follistatin supplementation induces changes in CDX2 CpG methylation and improves in vitro development of bovine SCNT preimplantation embryos.

Caudal Type Homeobox 2 (CDX2) is a key regulator of trophectoderm formation and maintenance in preimplantation embryos. We previously demonstrated that supplementation of exogenous follistatin, during in vitro culture of bovine IVF embryos, upregulates CDX2 expression, possibly, via alteration of the methylation status of CDX2 gene. Here, we further investigated the effects of exogenous follistatin supplementation on developmental competence and CDX2 methylation in bovine somatic cell nuclear transfer (SCNT) embryos. SCNT embryos were cultured with or without follistatin for 72h, then transferred into follistatin free media until d7 when blastocysts were collected and subjected to CDX2 gene expression and DNA methylation analysis for CDX2 regulatory regions by bisulfite sequencing. Follistatin supplementation significantly increased both blastocyst development as well as blastocyst CDX2 mRNA expression on d7. Three different CpG rich fragments within the CDX2 regulatory elements; proximal promoter (fragment P1, -1644 to -1180; P2, -305 to +126) and intron 1 (fragment I, + 3030 to + 3710) were identified and selected for bisulfite sequencing analysis. This analysis showed that follistatin treatment induced differential methylation (DM) at specific CpG sites within the analyzed fragments. Follistatin treatment elicited hypomethylation at six CpG sites at positions -1374, -279, -163, -23, +122 and +3558 and hypermethylation at two CpG sites at positions -243 and +20 in promoter region and first intron of CDX2 gene. Motif analysis using MatInspector revealed that differentially methylated CpG sites are putative binding sites for key transcription factors (TFs) known to regulate Cdx2 expression in mouse embryos and embryonic stem cells including OCT1, AP2F, KLF and P53, or TFs that have indirect link to CDX2 regulation including HAND and NRSF. Collectively, results of the present study together with our previous findings in IVF embryos support the hypothesis that alteration of CDX2 methylation is one of the epigenetic mechanisms by which follistatin may regulates CDX2 expression in preimplantation bovine embryos.

Choline acts during preimplantation development of the bovine embryo to program postnatal growth and alter muscle DNA methylation.

The preimplantation period of embryonic development can be a key window for programming of postnatal development because extensive epigenetic remodeling occurs during this time. It was hypothesized that modification of one-carbon metabolism of the bovine embryo by addition of the methyl-donor choline to culture medium would change postnatal phenotype through epigenetic modification. Embryos produced in vitro were cultured with 1.8 mM choline chloride or control medium. Blastocysts were transferred into females and pregnancy outcomes and postnatal phenotype of the resultant calves determined. Exposure of embryos to choline increased gestation length and calf birth weight. Calves derived from choline-treated embryos were also heavier at weaning and had increased ratio of body weight to hip height than control calves. Choline altered muscle DNA methylation of calves 4 months after birth. A total of 670 of the 8149 CpG examined were differentially methylated, with the predominant effect of choline being hypomethylation. Among the genes associated with differentially methylated CpG were ribosomal RNAs and genes in AMPK, mTOR, integrin, and BEX2 canonical pathways and cellular functions involved in growth and proliferation. Results demonstrate that provision of the methyl-donor choline to the preimplantation embryo can alter its developmental program to increase gestation length, birth weight, and weaning weight and cause postnatal changes in muscle DNA methylation including those associated with genes related to anabolic processes and cellular growth. The importance of the nutritional status of the embryo with respect to one-carbon metabolism for ensuring health and well-being after birth is emphasized by these observations.

Comparing mRNA and sncRNA profiles during the maternal-to-embryonic transition in bovine IVF and scNT embryos†.

Production of embryos with high developmental competence by somatic cell nuclear transfer (scNT) is far less efficient than for in vitro fertilized (IVF) embryos, likely due to an accumulation of errors in genome reprogramming that results in aberrant expression of RNA transcripts, including messenger RNAs (mRNA) and, possibly, microRNAs (miRNA). Thus, our objectives were to use RNAseq to determine the dynamics of mRNA expression in early developing scNT and IVF embryos in the context of the maternal-to-embryonic transition (MET) and to correlate apparent transcriptional dysregulation in cloned embryos with miRNA expression profiles. Comparisons between scNT and IVF embryos indicated large scale transcriptome differences, which were most evident at the 8-cell and morula stages for genes associated with biological functions critical for the MET. For two miRNAs previously identified as differentially expressed in scNT morulae, miR-34a and miR-345, negative correlations with some predicted mRNA targets were apparent, though not widespread among the majority of predicted targets. Moreover, although large-scale aberrations in expression of mRNAs were evident during the MET in cattle scNT embryos, these changes were not consistently correlated with aberrations in miRNA expression at the same developmental stage, suggesting that other mechanisms controlling gene expression may be involved.

Impact of heat stress on embryonic development during first 16 days of gestation in dairy cows.

Objective was to elucidate the effects of heat stress (HS) on embryo development during first 16 gestational days (GD) and circulating hormone concentrations on GD-16 in lactating Holstein cows. Cows in HS and control (CON) groups were exposed to temperature humidity index (THI) of ≥ 73 and < 73, respectively, for 3 weeks before the experiment. GD-7 (67 vs 49%) and GD-16 (52 vs. 31%) conception rates following single insemination were greater (P < 0.01) for CON compared with HS cows. Control cows produced more GD-7 transferrable embryos following superovulation compared with HS cows (84.8 vs 53.1%; P < 0.001). Mean (± SEM) length (45.2 ± 10.6 vs. 59.2 ± 9.1 mm) and weight (31.4 ± 4.3 vs. 42.4 ± 6.2 mg) of GD-16 conceptus were greater for CON compared with HS cows (P < 0.05). Control cows yielded more filamentous conceptus (≥ 25 mm) compared with HS cows (71 vs 45%; P < 0.05). Progesterone (2.09-fold) was higher, and cortisol (1.86-fold), prolactin (1.60-fold), substance-P (1.55-fold), Isoprostane-8 (1.34-fold) and prostaglandin F metabolites (1.97-fold) were lower in CON compared with HS cows (P < 0.05). Progesterone positively, and substance-P, isoprostane-8 and the THI negatively were associated with GD-16 conceptus length (P < 0.05). In conclusion, altered hormones concentrations in heat-stressed cows plausibly resulted in lower GD-7 and GD-16 conception rates, fewer GD-7 transferable embryos, and stunted GD-16 conceptus elongation.

Constitutive Expression of TERT Enhances ß-Klotho Expression and Improves Age-Related Deterioration in Early Bovine Embryos.

Age-associated decline in oocyte quality is one of the dominant factors of low fertility. Aging alters several key processes, such as telomere lengthening, cell senescence, and cellular longevity of granulosa cells surrounding oocyte. To investigate the age-dependent molecular changes, we examined the expression, localization, and correlation of telomerase reverse transcriptase (TERT) and ß-Klotho (KLB) in bovine granulosa cells, oocytes, and early embryos during the aging process. Herein, cumulus-oocyte complexes (COCs) obtained from aged cows (>120 months) via ovum pick-up (OPU) showed reduced expression of ß-Klotho and its co-receptor fibroblast growth factor receptor 1 (FGFR1). TERT plasmid injection into pronuclear zygotes not only markedly enhanced day-8 blastocysts' development competence (39.1 ± 0.8%) compared to the control (31.1 ± 0.5%) and D-galactose (17.9 ± 1.0%) treatment groups but also enhanced KLB and FGFR1 expression. In addition, plasmid-injected zygotes displayed a considerable enhancement in blastocyst quality and implantation potential. Cycloastragenol (CAG), an extract of saponins, stimulates telomerase enzymes and enhances KLB expression and alleviates age-related deterioration in cultured primary bovine granulosa cells. In conclusion, telomerase activation or constitutive expression will increase KLB expression and activate the FGFR1/ß-Klotho pathway in bovine granulosa cells and early embryos, inhibiting age-related malfunctioning.

Dynamics of small non-coding RNAs in bovine scNT embryos through the maternal-to-embryonic transition†.

The efficiency of somatic cell nuclear transfer (scNT) for production of viable offspring is relatively low as compared to in vitro fertilization (IVF), presumably due to deficiencies in epigenetic reprogramming of the donor cell genome. Such defects may also involve the population of small non-coding RNAs (sncRNAs), which are important during early embryonic development. The objective of this study was to examine dynamic changes in relative abundance of sncRNAs during the maternal-to-embryonic transition (MET) in bovine embryos produced by scNT as compared to IVF by using RNA sequencing. When comparing populations of miRNA in scNT versus IVF embryos, only miR-2340, miR-345, and miR34a were differentially expressed in morulae, though many more miRNAs were differentially expressed when comparing across developmental stages. Also of interest, distinct populations of piwi-interacting like RNAs (pilRNAs) were identified in bovine embryos prior to and during embryonic genome activation (EGA) as compared bovine embryos post-EGA and differentiated cells. Overall, sncRNA sequencing analysis of preimplantation embryos revealed largely similar profiles of sncRNAs for IVF and scNT embryos at the 2-cell, 8-cell, morula, and blastocyst stages of development. However, these sncRNA profiles, including miRNA, piRNA, and tRNA fragments, were notably distinct prior to and after completion of the MET.

Nutritional parameters in the blood of dams during late gestation and immediately after calving, in the umbilical vein at calving, and in the blood of calves immediately following birth in Holstein heifers pregnant with either Holstein or beef breed fetuses.

Dairy cattle management lacks consideration of fetal breed, the effect of which on fetal growth and nutrition are unclear. We investigated blood parameters in 12 late-pregnant Holstein heifers with similar (Holstein, n = 5) or different (Japanese Black [n = 4] or F1 cross [n = 3]; Holstein × Japanese Black) fetus breeds and in their umbilical cords and calves. Samples were obtained from dams 1 week before calving (-1 week) and immediately after calving, from the umbilical vein at calving, and from calves immediately after birth. Dams with beef fetuses had higher serum glucose levels (-1 week; p < .05) than those with Holstein fetuses. Plasma total amino acid, total essential amino acid, total nonessential amino acid, and other amino acid concentrations were lower in the umbilical veins of dams with calves of the beef breeds than in those of the Holstein breeds (p < .05). Furthermore, serum glucose and plasma amino acid levels were lower in the beef calves than in the Holstein calves (p < .05). Overall, nutrient supply from dams to beef fetuses was lower than that to Holstein fetuses. Our findings may facilitate feeding management of dairy cattle pregnant with beef breeds for appropriate fetal growth and nutrition.

Influence of estradiol on bovine trophectoderm and uterine gene transcripts around maternal recognition of pregnancy†.

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.

Maturation of bovine oocytes under low culture temperature decreased glutathione peroxidase activity of both oocytes and blastocysts.

It is known that the basic variable in the cellular environment is temperature and low temperature decreases cellular metabolism rate. Also, low cellular metabolic activity reduces oxidative stress, resulting in low ROS production. The aim of this study was therefore to investigate the effect of 36.5°C (low) and 38.5°C (conventional) incubation temperatures during IVM on glutathione peroxidase activity of oocytes and blastocysts following fertilization. Bovine oocytes were matured in medium-199 for 22 hours at either 36.5°C or 38.5°C and they were subjected to in vitro fertilization (IVF). Putative zygotes were then transferred randomly into SOFaa embryo culture media with or without antioxidant (a mixture of GSH and SOD) until development to the blastocyst stage. Glutathione peroxidase enzyme (GSH-Px) activity was lower (p⟨0.05) in oocytes matured at low temperature than those of conventional temperature. Similarly, GSH-Px activity was lower (p⟨0.05) in blastocysts, which were obtained from oocytes matured at low temperature and cultured in antioxidants-supplemented embryo media. The GSH-Px activity of blastocysts, obtained from oocytes matured in low temperature, cultured in antioxidants-free embryo media was similar to blastocysts obtained from oocytes matured in conventional temperature, cultured in antioxidants-supplemented embryo media. The results of the present study show that decreasing the in vitro maturation temperature decreases antioxidant enzyme activity in both oocyte and blastocyst. Additionally, maturation of bovine oocytes at 36.5°C incubation temperature may provide an optimal thermal condition for the enzymatic antioxidant system of both oocytes and blastocyst.

Production of a Cloned Offspring and CRISPR/Cas9 Genome Editing of Embryonic Fibroblasts in Cattle.

Somatic Cell Nuclear Transfer (SCNT) technique was used to produce the first viable cloned cattle offspring in Russia. Whole-genome SNP genotyping confirmed that the cloned calf was identical to the fibroblast cell line that was used for SCNT. CRISPR/Cas9 approach was subsequently used to knock out genes for beta-lactoglobulin gene (PAEP) and the beta-lactoglobulin-like protein gene (LOC100848610) in the fibroblast cells. Gene editing (GE) efficiency was 4.4% for each of these genes. We successfully obtained single-cell-derived fibroblast colonies containing PAEP and LOC100848610 knockouts, which will be used to produce beta-lactoglobulin-deficient cattle.

NOTCH signaling pathway is required for bovine early embryonic development†.

The NOTCH signaling pathway plays an important role in regulating various biological processes, including lineage specification and apoptosis. Multiple components of the NOTCH pathway have been identified in mammalian preimplantation embryos. However, the precise role of the NOTCH pathway in early embryonic development is poorly understood, especially in large animals. Here, we show that the expression of genes encoding key transcripts of the NOTCH pathway is dynamic throughout early embryonic development. We also confirm the presence of active NOTCH1 and RBPJ. By using pharmacological and RNA interference tools, we demonstrate that the NOTCH pathway is required for the proper development of bovine early embryos. This functional consequence could be partly attributed to the major transcriptional mediator, Recombination Signal Binding Protein For Immunoglobulin Kappa J Region (RBPJ), whose deficiency also compromised the embryo quality. Indeed, both NOTCH1 and RBPJ knockdown cause a significant increase of histone H3 serine 10 phosphorylation (pH3S10, a mitosis marker) positive blastomeres, suggesting a cell cycle arrest at mitosis. Importantly, RNA sequencing analyses reveal that either NOTCH1 or RBPJ depletion triggers a reduction in H1FOO that encodes the oocyte-specific linker histone H1 variant. Interestingly, depleting H1FOO results in detrimental effects on the developmental competence of early embryos, similar with NOTCH1 inhibition. Overall, our results reveal a crucial role for NOTCH pathway in regulating bovine preimplantation development, likely by controlling cell proliferation and maintaining H1FOO expression.

Promoter-specific expression of the imprinted IGF2 gene in bovine oocytes and pre-implantation embryos.

The bovine IGF2 locus is a genomic region with alternative transcripts controlled by five promoters (P0, P1, P2, P3 and P4). As transcriptional regulation can affect messenger RNA (mRNA) stability and translation, and thus, subsequent biological effects, this study evaluated the bovine IGF2 promoter-specific expression patterns in oocytes and pre-implantation embryos produced in vitro by our standard IVP procedures. Immature and matured oocytes, and pre-implantation embryos at the 1-, 2-, 4-, 8- and 16-cell, and at early morula, compact morula, blastocyst and expanded blastocyst stages were collected in three pools of five structures per stage, in four replicates. Total RNA was extracted and subjected to RT-qPCR, using four sets of IGF2 promoter-specific primers covering transcripts driven by promoters P0/P1, P2, P3 and P4, with fragments sequenced for confirmation. Expression of P2- and P4-derived transcripts showed an initial peak between immature (P4) or matured (P2/P4) oocytes and 2-cell embryos, gradually falling until embryo genome activation (EGA), rising again at compaction and cavitation. P0/P1-derived transcripts were identified after EGA, during compaction, whereas P3 activity was not detected at any stage. Our findings suggest that P0/P1 and P2 likely have secondary roles during early stages, whereas P3 may be more relevant later in development. P4 seems to be the main pathway for bovine IGF2 expression during oocyte maturation and embryo development and, therefore, the main target to influence IVP in modulation of embryo growth and in studies in developmental biology.

Effect of vitrification on global gene expression dynamics of bovine elongating embryos.

Embryo vitrification involves exposure to high concentrations of cryoprotectants and osmotic stress during cooling and warming in the cryopreservation process. Many of these factors can potentially affect gene expression. In this study, invitro-produced bovine embryos at the blastocyst stage were subjected to vitrification. Four recipients each were used for transferring non-vitrified (n=80) and vitrified (n=80) embryos. A total of 12 non-vitrified and 9 vitrified viable day-14 (D14) embryos were recovered by uterine flushing. RNA-seq analysis of the whole embryo or isolated trophectoderm (TE) from vitrified and fresh recovered D14 embryos revealed a total of 927 and 4376 genes with changed expression in embryos and TE isolates, respectively, as a result of vitrification. In addition, we found 671 and 61 genes commonly up- or downregulated in both vitrified whole embryos and TE. Commonly upregulated pathways by vitrification included epithelial adherens junctions, sirtuin signalling, germ cell-sertoli cell junction, ATM signalling, NER and protein ubiquitination pathways. The commonly downregulated pathways included EIF2 signalling, oxidative phosphorylation, mitochondrial dysfunction, regulation of eIF4 and p70S6K signalling and mTOR signalling pathways. Our analysis identified specific pathways and implicated specific gene expression patterns affecting embryo developmental competence that are important to cryopreservation.

Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1.

In vitro embryo production systems are limited by their inability to consistently produce embryos with the competency to develop to the blastocyst stage, survive cryopreservation, and establish a pregnancy. Previous work identified a combination of three cytokines [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1)], called FLI, that we hypothesize improve preimplantation development of bovine embryos in vitro. To test this hypothesis, FLI was supplemented into oocyte maturation or embryo culture medium. Embryos were produced in vitro using abattoir-derived oocytes and fertilized with sperm from a single bull known to have high fertility. After an 18-20 h fertilization period, putative zygotes were cultured in synthetic oviductal fluid (SOF) for 8 days. The addition of FLI to the oocyte maturation medium increased (P < 0.05) the dissociation of transzonal projections at 12, 18, and 24 h of maturation, as well as, the proportion of oocytes that reached the metaphase II stage of meiosis. Additionally, lipid content was decreased (P < 0.05) in the blastocyst stage embryo. The addition of FLI during the culture period increased development to the blastocyst stage, cytoskeleton integrity, and survival following slow freezing, as well as, decreased post thaw cell apoptosis (P < 0.05). In conclusion, the supplementation of these cytokines in vitro has the potential to alleviate some of the challenges associated with the cryo-survival of in vitro produced bovine embryos through improving embryo development and embryo quality.

Innate and humoral immune parameters at delivery in colostrum and calves from heifers experimentally infected with Neospora caninum.

Neospora caninum is a leading cause of abortion in cattle worldwide. The study of the immune response against N. caninum is critical to understand its epidemiology, pathogenesis, diagnosis and, ultimately, in preventing and controlling bovine neosporosis. Herein, we determined the gene expression of innate immune components endosomal RNA-sensing TLRs, BMAP28 cathelicidin, TNF-α and IL-10 and characterized the variation in both IgG ratio and avidity at delivery in N. caninum-infected heifers challenged at day 210 of gestation, colostrum and their calves. Increased BMAP28 expression was observed not only in colostrum but also in peripheral blood mononuclear cells (PBMC) and umbilical cord of calves from N. caninum-infected heifers in comparison with mock-infected control group. In addition, statistically significant decrease of TLR7 and IL-10 expression levels were observed in umbilical cord, suggesting an attempt to avoid an exacerbated immune response against the parasite. At delivery, serum and colostrum samples from infected group evidenced specific IgG anti-N. caninum. Infected heifers showed IgG1/IgG2 ratios <1 and high avidity specific IgG. As expected, colostrum samples of these animals exhibited a high IgG1 concentration and elevated avidity values. Three out of four calves from N. caninum-infected heifers had specific IgG with IgG1/IgG2 ratios>1 and lower avidity values before colostrum intake. Interestingly, both IgG1/IgG2 ratios and avidity values increased in seropositive calves after colostrum intake. Overall, this study provides novel information on neonatal immunity in congenitally infected calves, which is essential to understand how the immune pathways could be manipulated or immune components could be employed in order to improve protection against neosporosis.

Evaluation of a small volume oil-free in vitro production system for bovine embryos.

BACKGROUND: Embryos are usually produced in culture systems with an oil overlay, which conveys protection against the evaporation of water and microbial contamination. The oil can also release toxic substances and absorb essential components, such as hormones, which adversely affect the quality of the oocytes and the development of embryos in vitro. OBJECTIVE: The aim of this study was to validate an oil-free bovine in vitro production (IVP) system. METHOD: Cumulus-oocyte complexes collected from abattoir-derived ovaries were matured, fertilized and cultured employing a standard system. The quantity of medium in both groups (with and without an oil overlay) and throughout all stages of IVP was maintained at a volume of 100 µl. The oil group was covered with paraffin oil. The maturation stage of oocytes was assessed using fluorescence staining after 24 hr and developmental stages of embryos were evaluated on day 8. The expanded day 8 blastocysts were assessed by live-dead staining. RESULTS: Oocytes matured in the absence of an oil overlay had significantly higher maturation rates when compared against matured oocytes in medium with an oil overlay. Steroid concentration is higher in medium after maturation without oil cover. The developmental rate was significantly higher after culture without oil overlay. The total cell number and the live-dead ratio was not significantly different. The osmolality did not differ between both groups during maturation and slightly decreased during culture without oil. CONCLUSION: Based on the current study, bovine oil-free IVP systems can be suggested as an alternative to oil-covered medium.

Analysis of novel embryonic factors of cattle and effects on endometrial cells in vitro.

Interferon tau (IFNT) is thought to have essential functions in maternal recognition and establishment of pregnancy in ruminants. There, however, is a lack of research on embryonic factors that affect pregnancy other than IFNT. The present study was conducted to determine what are other embryo-derived factors involved in pregnancy recognition and to identify effects on endometrial cells using an in vitro culture system. With use of LC-MS/MS procedures to evaluate the supernatant of elongating embryos of cattle in culture, there was detection of 78 secretary proteins including five cytokines and two growth factors. Then there was analysis for up-regulated genes using ingenuity pathway procedure, IFNT and MIF were identified as upstream regulators of 37 and five genes, respectively. The mRNA transcript of MIF receptors was identified in endometrial cells, however, not in embryos. Among genes induced by MIF, CCL2, IL7 and IL23A transcripts were identified in endometrial cells. When endometrial cells were treated with interferon alpha (IFNA) and MIF, the CCL2 transcript was in a larger abundance of endometrial epithelial and polymorphonuclear neutrophil cells, and there was a larger abundance of there mRNA transcripts as a result of MIF treatment of peripheral blood mononuclear cells. In conclusion, MIF secreted by elongating embryos of cattle synergistically regulates relative abundances of specific mRNA transcripts of endometrial cells when there is treatment with IFNA, indicating further there are several factors other than IFNT that have effects on gene expression in the endometrium during early stages of gestation in cattle.

Effect of types of breeding on embryo survival following first AI in lactating Holstein cows.

The main objective was to investigate the effects of timed-AI protocols versus AI following oestrus detection on circulating progesterone (P4) and embryo survival after first service in Holstein cows. Cycling status was determined by ultrasonography and by plasma P4 concentrations 14 and 26 days after calving, and only cows with a corpus luteum and/or P4 ≥ 1 ng/ml were used. Cows were randomly allocated to one of three types of breeding: DO (n = 80), received GnRH-7d-PGF2α-3d-GnRH and Ovsynch56 was initiated 7 days later; G7G (n = 70), received PGF2α-2d-GnRH and Ovsynch56 (GnRH-7d-PGF2α-56h-GnRH-16h-AI) was initiated 7 days later; or AI based on oestrus detection, EDAI (n = 60). Progesterone was also determined at AI and 8, 16, 18 and 20 days after AI; ISG15 and MX2 mRNA abundance were determined 16 days after AI. Mean plasma P4 at AI was greater in the EDAI group compared with DO and G7G groups, while after AI, P4 was greater in DO and G7G groups compared with EDAI group. However, the percentage of cows with a concentration of P4 < 0.8 ng/ml at AI did not differ among groups. Relative mRNA abundance of ISG15 and MX2 was greater in the DO and G7G groups compared to those in EDAI group. Pregnancy per AI 16, 32 and 60 days after AI was greater (p < .05) in cows in the DO group compared with those in EDAI group (47.5%, 38.8% and 36.3% vs. 30.0%, 21.7% and 15.0%). Pregnancy losses between 16 and 60 days after AI were greater (p < .05) in cows in the EDAI (50.0%) group compared to those subjected to DO (23.7%) or G7G (24.1%). In conclusion, the use of timed-AI synchronization protocols resulted in greater circulating P4 concentrations post-AI and greater embryo survival following first service in lactating Holstein cows.

Alternate PCR assays for screening of JH1 mutation associated with embryonic death in Jersey cattle.

Jersey haplotype (JH) 1, a stop-gain lethal mutation in the CWC15 gene, causes embryonic losses in Jersey cattle. Two PCR based assays using Amplification Refractory Mutation System (T-ARMS-PCR) and restriction fragment length polymorphism (PCR-RFLP) were developed for screening of the JH1 in cattle. During the screening, seven among 30 Indian Jersey bulls were identified as carriers of the mutant JH1 allele, the first time in the country. These PCR assays are economical, rapid and accurate; and can be used separately or in combination for screening and cross-validation of the JH1 carriers in Jersey cattle.