Effects of choline on the phenotype of the cultured bovine preimplantation embryo.

Choline is a precursor of acetylcholine, phosphatidylcholine, and the methyl-donor betaine. Reports indicate that supplementation with rumen-protected choline improves postpartum reproductive function of dairy cows. The objective was to determine whether addition of choline to culture medium of in vitro-produced embryos alters the phenotype of the resultant blastocysts. Treatments were choline chloride (ChCl; 0.004, 1.3, 1.8, and 6.37 mM) and phosphatidylcholine (1.3 mM). Treatment with 0.004 mM ChCl improved development to the blastocyst stage, increased blastocyst cell number, and increased the percentage of blastocysts that were hatching or hatched. Development was not affected by higher concentrations of ChCl but was reduced by 1.3 mM phosphatidylcholine. Treatment of embryos with 1.3 mM ChCl (but not other concentrations) increased expression in blastocysts of 11 of 165 genes examined (AMOT, NANOG, HDAC8, HNF4A, STAT1, MBNL3, SOX2, STAT3, KDM2B, SAV1, and GPAM) and decreased expression of one gene (ASS1). Treatment with 1.3 mM ChCl decreased global DNA methylation at d 3.5 of development and increased DNA methylation at d 7.5 in blastocysts. Treatment with 1.8 mM ChCl also increased methylation in blastocysts. In conclusion, addition of choline to the culture medium alters the phenotype of preimplantation bovine embryos produced in vitro. Choline chloride can act in a concentration-dependent manner to alter development, expression of specific genes, and DNA methylation.

Molecular fingerprint of female bovine embryos produced in vitro with high competence to establish and maintain pregnancy†.

The objective was to identify the transcriptomic profile of in vitro-derived embryos with high competence to establish and maintain gestation. Embryos produced with X-sorted sperm were cultured from day 5 to day 7 in serum-free medium containing 10 ng/ml recombinant bovine colony-stimulating factor 2 (CSF2) or vehicle. The CSF2 was administered because this molecule can increase blastocyst competence for survival after embryo transfer. Blastocysts were harvested on day 7 of culture and manually bisected. One demi-embryo from a single blastocyst was transferred into a synchronized recipient and the other half was used for RNA-seq analysis. Using P < 0.01 and a fold change >2-fold or <0.5 fold as cutoffs, there were 617 differentially expressed genes (DEG) between embryos that survived to day 30 of gestation vs those that did not, 470 DEG between embryos that survived to day 60 and those that did not, 432 DEG between embryos that maintained pregnancy from day 30 to day 60 vs those where pregnancy failed after day 30, and 635 DEG regulated by CSF2. Pathways and ontologies in which DEG were overrepresented included many related to cellular responses to stress and cell survival. It was concluded that gene expression in the blastocyst is different between embryos that are competent to establish and maintain pregnancy vs those that are not. The relationship between expression of genes related to cell stress and subsequent embryonic survival probably reflects cellular perturbations caused by embryonic development taking place in the artificial environment associated with cell culture.

Follicular fluid exosomes act on the bovine oocyte to improve oocyte competence to support development and survival to heat shock.

Addition of follicular fluid to oocyte maturation medium can affect cumulus cell function, increase competence of the oocytes to be fertilised and develop to the blastocyst stage and protect the oocyte from heat shock. Here, it was tested whether exosomes in follicular fluid are responsible for the effects of follicular fluid on the function of the cumulus-oocyte complex (COC). This was accomplished by culturing COCs during oocyte maturation at 38.5°C (body temperature of the cow) or 41°C (heat shock) with follicular fluid or exosomes derived from follicular fluid and evaluating various aspects of function of the oocyte and the embryo derived from it. Negative effects of heat shock on cleavage and blastocyst development, but not cumulus expansion, were reduced by follicular fluid and exosomes. The results support the idea that exosomes in follicular fluid play important roles during oocyte maturation to enhance oocyte function and protect it from stress.

Identification of potential embryokines in the bovine reproductive tract.

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the preimplantation embryo is largely incomplete. The goal of the present experiment was to identify candidates for this function. The approach was to assess expression patterns in the endometrium and oviduct of 93 genes encoding for hormones, growth factors, chemokines, cytokines, and WNT-related molecules. Results show that all of the genes were expressed in the reproductive tract. Expression in oviduct was affected by day of the estrous cycle for 21 genes with 11 genes having highest expression at estrus (CCL21, CTGF, CXCL10, CXCL16, DKK3, FGF10, IL18, IL33, IL34, PGF, and SFRP2), 1 gene at d 3 (WNT4), 8 at d 5 (BMP7, HGF, IL6, SFRP1, TGFB1, WIF1, WNT2, and WNT5A), and 1 at d 7 (IK). For endometrium, expression of 34 genes was affected by day of the estrous cycle with 11 having highest expression at d 0 (BMP7, CCL14, CCL21, CCL26, CTGF, CXCL12, IGF2, IL16, IL33, SFRP2, and WIF1), 2 at d 3 (HDGF, IL15), 14 at d 5 (CSF2, CX3CL1, CXCL3, FGF1, FGF2, GRO1, HGF, IGF1, IL1B, IL8, SFRP1, SFRP4, WNT5A, and WNT16), and 7 at d 7 (CXCL16, FGF13, HDGFRP2, TDGF1, VEGFB, WNT7A, and WNT11). Results are consistent with a set of genes regulated by estradiol early in the estrous cycle and another set regulated by progesterone later in the cycle. The cell-signaling genes identified here as being expressed in the oviduct and endometrium could serve to regulate early embryonic development in a stage-of-pregnancy-specific manner.

Consequences of exposure of embryos produced in vitro in a serum-containing medium to dickkopf-related protein 1 and colony stimulating factor 2 on blastocyst yield, pregnancy rate, and birth weight.

Embryokines are molecules secreted by the mother that regulate embryonic development. Among these molecules in cattle are colony stimulating factor 2 (CSF2) and dickkopf-related protein 1 (DKK1). Here, we evaluated actions of CSF2 and DKK1 alone or in combination on characteristics of embryos produced in vitro in the presence of serum. A total of 70 beef cows from 4 farms were subjected to oocyte retrieval on 1 to 4 occasions. Within each farm, donors were randomly allocated to 1 of 4 treatment groups (vehicle, CSF2, DKK1, CSF2 + DKK1). Embryos from a given donor were always exposed to the same treatment. Treatments were added to the culture medium on d 5 after insemination, and blastocyst stage embryos were transferred to recipient females 2 d later. Treatment did not affect the percent of oocytes or cleaved embryos that developed to the blastocyst stage or the percent of recipients that became pregnant after embryo transfer. However, calves derived from embryos treated with DKK1 were smaller at birth, regardless of CSF2 treatment. Results indicate no effects of addition of CSF2 or DKK1 to culture of embryos produced in vitro with serum-containing medium on development to the blastocyst stage or competence to establish pregnancy after transfer to recipients. The fact that embryos cultured with DKK1 resulted in calves with reduced birth weight illustrates the potential ability of this embryokine to program postnatal phenotype. Results support the concept that properties of the offspring can be programmed as early as the preimplantation period.

Effects of rumen-protected methionine and choline supplementation on the preimplantation embryo in Holstein cows.

Our objective was to determine the effects of supplementing methionine and choline during the prepartum and postpartum periods on preimplantation embryos of Holstein cows. Multiparous cows were assigned in a randomized complete-block design into four treatments from 21 days before calving to 30 days in milk (DIM). Treatments (TRT) were MET (n = 9, fed the basal diet + rumen-protected methionine at a rate of 0.08% [w:w] of the dry matter [DM], Smartamine M), CHO (n = 8, fed the basal diet + choline 60 g/d, Reashure), MIX (n = 11, fed the basal diet + Smartamine M and 60 g/d Reashure), and CON (n = 8, no supplementation, fed the close-up and fresh cow diets). Cows were randomly reassigned to two new groups (GRP) to receive the following diets from 31 to 72 DIM; control (CNT, n = 16, fed a basal diet) and SMT (n = 20, fed the basal diet + 0.08% [w:w] of the dry matter intake as methionine). An progesterone intravaginal insert (CIDR) device was inserted in all cows after follicular aspiration (60 DIM) and superovulation began at Day 61.5 using FSH in eight decreasing doses at 12-hour intervals over a 4-day period. On Days 63 and 64, all cows received two injections of PGF2α, and CIDR was removed on Day 65. Twenty-four hours after CIDR removal, ovulation was induced with GnRH. Cows received artificial insemination at 12 hours and 24 hours after GnRH. Embryos were flushed 6.5 days after artificial insemination. Global methylation of the embryos was assessed by immunofluorescent labeling of 5-methylcytosine, whereas lipid content was assessed by staining with Nile red. Nuclear staining was used to count the total number of cells per embryo. There was no difference between TRT, GRP, or their interaction (P > 0.05) for embryo recovery, embryos recovered, embryo quality, embryo stage, or cells per embryo. Methylation of the DNA had a TRT by GRP interaction (P = 0.01). Embryos from cows in CON-CNT had greater (P = 0.04) methylation (0.87 ± 0.09 arbitrary units [AU]) than embryos from cows in MET-CNT (0.44 ± 0.07 AU). The cytoplasmic lipid content was not affected (P > 0.05) by TRT or their interaction, but lipid content was greater (P = 0.04) for SMT (7.02 ± 1.03 AU) than that in CNT (3.61 ± 1.20 AU). In conclusion, cows in MET-CNT had embryos with lower methylation, and SMT cows had a higher lipid content than CNT. Methionine supplementation seems to impact the preimplantation embryo in a way that enhances its capacity for survival because there is strong evidence that endogenous lipid reserves serve as an energy substrate.

Bioactivity of ovulation inducing factor (or nerve growth factor) in bovine seminal plasma and its effects on ovarian function in cattle.

To understand the role of ovulation-inducing factor (or nerve growth factor) (OIF [NGF]) in bovine seminal plasma, we (1) used an in vivo llama bioassay to test the hypothesis that bovine seminal plasma induces ovulation and CL development in llamas similar to that of llama seminal plasma when the dose of seminal plasma is adjusted to ovulation-inducing factor content (experiment 1) and (2) determined the effect of bovine seminal plasma on the interval to ovulation and luteal development in heifers (experiment 2). Within species, seminal plasma was pooled (n = 160 bulls, n = 4 llamas), and the volume of seminal plasma used for treatment was adjusted to a total dose of 250 µg of ovulation-inducing factor. In experiment 1, mature female llamas were assigned randomly to four groups and treated intramuscularly with either 10 mL of PBS (negative control, n = 5), 50-µg GnRH (positive control, n = 5), 6-mL of llama seminal plasma (n = 6), or 12 mL of bull seminal plasma (n = 6). Ovulation and CL development were monitored by transrectal ultrasonography. In experiment 2, beef heifers were given a luteolytic dose of prostaglandin followed by 25-mg porcine LH (pLH) 12 hours later to induce ovulation. Heifers were assigned randomly to three groups and given 12 mL bovine seminal plasma intramuscularly 12 hours after pLH treatment (n = 10), within 4 hours after ovulation (n = 9), or no treatment (control, n = 10). Ovulation was monitored by ultrasonography every 4 hours, and the CL development was monitored daily until the next ovulation. In experiment 1, ovulation was detected in 0/5, 4/5, 4/6, 4/6 llamas in the PBS, GnRH, llama seminal plasma, and bovine seminal plasma groups, respectively (P < 0.05). Luteal development was not different among groups. In experiment 2, the interval to ovulation was more synchronous (range: 4 vs. 22 hours; P < 0.0001) in heifers treated with seminal plasma before ovulation compared with the other groups. Luteal development was not different among groups; however, plasma progesterone concentrations tended to be greater in the postovulation treatment group compared with other groups. In summary, results confirmed the presence of bioactive ovulation-inducing factor in bull seminal plasma and supported the hypothesis that bovine and llama seminal plasma have similar ovulatory effects, using a llama bioassay. Treatment with bovine seminal plasma resulted in greater synchrony of ovulation in heifers pretreated with pLH. Plasma progesterone concentration tended to be higher in heifers given bovine seminal plasma within 4 hours after ovulation, suggesting that bovine ovulation-inducing factor is luteotrophic.