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The slick-hair phenotype in cattle is due to one of a series of mutations in the prolactin receptor (PRLR) that cause truncation of the C-terminal region of the protein involved in JAK2/STAT5 activation during prolactin signaling. Here we evaluated whether the inheritance of the SLICK1 allele, the first slick mutation discovered, is inherited in a fashion consistent with Hardy-Weinberg equilibrium. It was hypothesized that any deleterious effect of inheriting the allele on embryonic or fetal function would result in reduced frequency of the allele in offspring. A total of 525 Holstein and Senepol cattle produced from matings involving one or both parents with the SLICK1 allele were genotyped. The observed frequency of the SLICK1 allele (0.247) was not significantly different than the expected frequency of 0.269. These results support the idea that inheritance of the SLICK1 allele does not act in the embryo or fetus to modify its competence to complete development to term.

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.

Once it enters the uterus at d 4 to 5 after ovulation, the preimplantation bovine embryo is controlled in its development by regulatory signaling molecules from the mother called embryokines. Here, several cell-signaling molecules whose genes are expressed in the endometrium during d 5 to 7 after estrus were tested for the ability to affect the competence of the embryo for further development and the characteristics of the resultant blastocysts. Molecules tested were C-natriuretic peptide (CNP), IL-8, bovine morphogenetic protein 4 (BMP-4), IL-6, and leukemia inhibitory factor (LIF). None of the cell-signaling molecules tested improved the competence of the embryo to become a blastocyst; in fact, BMP-4 decreased development. All molecules modified attributes of the blastocyst formed in culture. In particular, CNP increased the number of cells in the ICM, whereas IL-8 decreased inner cell mass cell numbers and tended to increase the proportion of blastocysts that were hatching or hatched. In addition, BMP-4 decreased the proportion of blastocysts that were hatching. Interleukin-6 and, to a lesser extent, LIF activated the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in the inner cell mass, and LIF increased the percent of cells in the blastocyst that were positive for both NANOG and phosphorylated (activated) STAT3. In conclusion, our results indicate that CNP, IL-8, IL-6, LIF, and BMP-4 can modify embryonic development of the cow in a manner that affects characteristics of the resultant blastocyst. Further research is required to understand how these changes in characteristics of the blastocyst would affect competence of the embryo to establish and maintain pregnancy.

Colony-stimulating factor 3 (CSF3), also known as granulocyte colony-stimulating factor, is used to reduce the incidence of mastitis in cattle. Here, we tested whether recombinant bovine CSF3 at 1, 10, or 100 ng/mL acts on the bovine oocyte during maturation or on the developing embryo to modify competence for development and characteristics of the resultant blastocyst. For experiment 1, oocytes were matured with or without CSF3. The resultant embryos were cultured in a serum-free medium for 7.5 d. There was no effect of CSF3 on cleavage or on development to the blastocyst stage except that 100 ng/mL reduced the percent of putative zygotes and cleaved embryos becoming blastocysts. Expression of transcripts for 93 genes in blastocysts was evaluated by RT-PCR using the Fluidigm platform. Transcript abundance was affected by one or more concentrations of CSF3 for four genes only (CYP11A1, NOTCH2, RAC1, and YAP1). For experiment 2, cumulus-oocyte complexes (COC) were fertilized with either X- or Y-sorted semen. Putative zygotes were cultured in medium containing CSF3 treatments added at the beginning of culture. There was no effect of CSF3, sex, or the interaction on the percent of putative zygotes that cleaved or on the percent of putative zygotes or cleaved embryos becoming a blastocyst. For experiment 3, CSF3 was added from day 4 to 7.5 of development. There was no effect of CSF3 on development to the blastocyst stage. Transcript abundance of 10 genes was increased by 100 ng/mL CSF3, including markers of epiblast (NANOG, SOX2), hypoblast (ALPL, FN1, KDM2B, and PDGFRA), epiblast and hypoblast (HNF4A) and trophectoderm (TJAP1). Results are indicative that concentrations of CSF3 higher than typical after therapeutic administration can reduce oocyte competence and act on the embryo to affect characteristics of the blastocyst.

A protocol for production of bovine embryos from oocytes collected from ovaries obtained from an abattoir is described. The protocol includes methods for in vitro maturation of oocytes, capacitation of sperm, fertilization, and development of the resultant embryos to the blastocyst stage. The protocol can be easily modified to use oocytes collected by ultrasound-guided follicular aspiration.

A protocol for production of bovine embryos from oocytes collected from ovaries obtained from an abattoir is described. The protocol includes methods for in vitro maturation of oocytes, capacitation of sperm, fertilization, and development of the resultant embryos to the blastocyst stage. The protocol can be easily modified to use oocytes collected by ultrasound-guided follicular aspiration.