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Establishing cell lines is a good model for experimental applications to study molecular mechanisms and cell-specific gene expression. Equids have a diffuse epitheliochorial placenta, where the invasive trophoblast is represented by the chorionic girdle (CG) and the noninvasive trophoblast by the allantochorion (AC). Embryonic CG cells are unique to horses and have a crucial role in equine chorionic gonadotropin (eCG) production and maintenance of pregnancy during the first trimester. This study had three objectives: (1) establishing a stable cell line from Day 30 CG cells and AC using lentivirus encoding hTERT; (2) characterisation of Day 30 CG cells and AC cell morphology and expression of eCG α (eCGA) and β (eCGB) subunits, major histocompatibility complex class II (MHCII), and Kisspeptin receptor (KISS1R) in CG and AC cells; (3) investigating eCG protein production invitro from Day 30 CG and AC cells. Three mares (n=3) were used to collect Day 30 conceptuses by non-surgical uterine lavage on Day 30 of pregnancy. All 3 conceptuses were dissected for CG and AC cells then cultured invitro to confluency in cell culture plates. Second-generation lentiviral particles were generated using a three-vector system including transfer vector pLV-hTERT-IRES-hygro, and human telomerase reverse transcriptase (hTERT) lentivirus was utilised to establish stable hygromycin-resistant equine embryonic cell lines. Reverse-transcription PCR (RT-PCR) was used to study gene expression in cells and radioimmunoassay was used to investigate protein presence in the media. We established a hygromycin-resistant Day 30 CG and AC cell lines that express eCGA, eCGB, and hTERT and confirmed using RT-PCR yielding the predicted bands. The cell lines were maintained for 16 passages (7±2 days/passage), 10 of which were cultured after the lentiviral infection steps. Also, we characterised CG cells as fast-growing, large, binucleated, and epithelioid, and AC cells as rapid-growing showing smaller, squamous, mononucleate, epithelioid, and elongated fibroblastic cells. The RT-PCR results showed eCGA and eCGB subunits are expressed by both Day 30 CG and AC cells, but MHCII and KISS1R genes were not expressed in either of cells. Moreover, radioimmunoassay results showed that Day 30 CG cells did produce eCG protein (35.42ngmL−1) invitro earlier than what previous literature has shown. However, Day 30 AC cells did not produce eCG protein (0.042ngmL−1) invitro, and both CG and AC cell lines stopped secreting eCG in the media after the lentiviral infection. To conclude, establishing stable and hygromycin-resistant cell lines from Day 30 equine CG and AC cells using lentivirus encoding pLV-hTERT-IRES-hygro is attainable. Also, equine chorionic gonadotropin eCG protein is produced invitro as early as Day 30 from CG cells.

MicroRNAs (miRNAs) are small, non-coding RNAs that are differentially expressed throughout the body, including the endometrium and chorion during gestation. Although they are produced by individual tissues, many of these miRNAs are excreted into circulation, either within exosomes or attached to protein carrier molecules. Circulating miRNAs are surprisingly stable, with an average half-life of 5 days. These characteristics suggest that circulating miRNAs have the potential to be powerful biomarkers following the characterisation of the normal population of miRNAs during pregnancy. Additionally, evaluation techniques must be considered, as some miRNAs are extremely sensitive to varying collection and analysis techniques. To evaluate the population of miRNAs in both pregnant and nonpregnant mares, whole blood was collected from mares at 10 months of gestation (n = 3), as well as at 9 days of diestrus (n = 3 for microarray; n = 4 for qPCR). Blood was collected using PAXgene blood miRNA kits (Qiagen, Valencia, CA, USA), and then stored at –20°C until use. RNA extraction was performed following the manufacturer’s instructions. Two different analysis platforms were compared. Quantitative RT-PCR (Lightcycler 480; Roche, Indianapolis, IN, USA) was used to evaluate 346 distinct miRNAs, whereas microarray (LC Sciences, Houston, TX, USA) was used to evaluate 275 miRNAs. In total, 222 of these miRNAs were analysed on both platforms for comparison purposes. Because of the large number of miRNAs evaluated, the robust response screening analysis method (JMP ver. 11.1; SAS Institute Inc., Cary, NC, USA) was used, and multiple comparisons were based upon a false discovery rate of P

Synchronization of oestrus in mares remains a challenge to practitioners using Assisted Reproductive Technologies. The research objective was to compare adverse reactions and reproductive parameters in mares treated with different sources of progestins for oestrus synchronization. Mixed breeds of mares with a mean age of 3 years (range 2–20) were used. Groups were: 1) altrenogest 0.044 mg kg–1 BW PO daily for 10 days (D) (Regumate, Merck, White House Station, NJ, USA) (n = 30), 2) Long-acting progesterone (LAP4) (BET Pharmacy, Lexington, KY, USA) (n = 30) 10 cc IM once in the neck, and 3) Controlled intravaginal drug release (CIDR-B 1.9 g P4; Zoetis, Kirkland, QC, Canada) (n = 15) vaginal insert for 10 days. Mares were randomly assigned to treatment and evaluated using transrectal reproductive ultrasonography on Days 1 (treatment initiation), 5, 10, and daily during oestrus until ovulation (Ov). On Day 10, mares were given 250 µg of cloprostenol (PG) (Estrumate, Merck) IM, and when a follicle (F) >35 mm was detected 2500 IU hCG (Chorulon, Intervet, Millsboro, MD, USA) was administered IM. Adverse reactions were scored as follows: Regumate – any reaction at any time; LAP4 – the injection site was inspected and scored as: 0, no reaction; 1, mild slight raised area; 2, moderate reaction 5 cm; 3, severe reaction 6–10 cm; 4, very severe reaction >10 cm on Day 5 post-injection; CIDR categorical scores were: 0, no discharge; 1, mild vaginal discharge on CIDR at withdrawal; 2, moderate discharge on tail or vulva; 3, severe urine scald or visible discharge; 4, very severe thickened inflamed skin from urine scald or discharge. Chi-squared test at P

Equine semen is commonly frozen in 0.5-mL straws at a concentration of 400 million spermatozoa per mL. Equine pregnancies have been produced in the past few years using assisted reproduction techniques that require very few sperm, such as sperm injection and gamete intrafallopian transfer. Consequently, thawing even a single straw would waste a high percentage of the sperm. The goals of this study were to: (1) evaluate the motility and viability of cryopreserved spermatozoa that had been thawed and refrozen, and (2) compare the effect of serial dilution on refreezing parameters. It was hypothesized that both motility and viability would be significantly decreased as a consequence of refreezing, but sufficient live, motile spermatozoa would be present for assisted reproductive techniques. Semen from six stallions of mixed light horse breeds was collected, evaluated, and frozen in lactose-EDTA in 0.5-mL straws. Semen from each stallion was subsequently thawed, held at room temperature for 10 minutes, and then refrozen in 0.25-mL straws. Additional semen from the initial freeze was thawed, diluted in the original extender, and refrozen at concentrations of 40×106, 4×106, 4× 105, and 4×104 spermatozoa per mL. Refrozen semen was evaluated for motility visually and by CASA. Viability of refrozen semen was determined by flow cytometry following staining with propidium iodide (PI). Percentages of motile spermatozoa prior to freezing, after the first freezing, and after the second freezing were compared using the PROC GLM model in SAS. All values are presented as the mean±standard deviation. Total motility decreased sigificantly (P0.05) subsequent motility or viability parameters. This study demonstrated that refrozen equine semen retained approximately 70% of the initial post-thaw motility. Dilution prior to refreezing would allow for the reallocation of a single 0.5-mL straw into hundreds or thousands of smaller straws for future use in assisted reproduction. Refreezing would allow for the judicious use of valuable stallion semen in limited supply.