113 PRELIMINARY CHARACTERIZATION OF OXYTOCINASE IN EQUINE SERUM

Abstract

Oxytocinase/insulin regulated aminopeptidase (IRAP) or leucyl-cystinyl aminopeptidase (LNPEP) is an enzyme that is involved in the regulation of hormones such as oxytocin, vasopressin, and angiotensin in both humans and sheep. Historically, very low levels of this aminopeptidase were reported in monthly samples obtained from cycling and pregnant mares using an enzymatic colourimetric method. The regulation of oxytocin in horses is of interest because of its central role in uterine clearance, luteal maintenance, parturition, passage of fetal membranes, maternal foal bonding, and milk let-down. A preliminary study was performed with the objective of re-examining the level of serum oxytocinase in nonpregnant control (n = 3 mares sampled every other day; EOD of the oestrous cycle), and n = 5 mares sampled Day 12 to 15; oxytocin-treated (n = 2 mares sampled Day 12 to 15), and early pregnant mares (n = 6 mares sampled EOD), using more sensitive ELISA methodology. Mares were examined daily in oestrus until ovulation (Day 0) and from Days 10 to 21, using transrectal ultrasonography of the reproductive tract. Palpable changes in uterine and cervical tone, ultrasound measurement of dominant follicles, oedema scores (0 to 4 with 4 being maximal oedema), and changes in luteal echotexture and size were recorded. Pregnant mares were bred using AI (>200 million motile and normal sperm) from a proven stallion while in oestrus until ovulation, beginning when the dominant follicle was >35 mm. Oxytocin-treated mares were administered 60 IU IM oxytocin SID from Day 7 to 14. Blood was collected to obtain serum. Changes in serum oxytocinase levels were measured using a commercially available ELISA kit for Horse LNPEP according to the manufacturer's instructions (MyBioSource, San Diego, CA, USA) and validated for use in our laboratory using serial dilutions of pooled serum with an intra-assay and inter-assay CV <15%. The lowest standard of the assay was 31.2 ng mL^–1. Preliminary results of this pilot study demonstrated in control cycles median serum oxytocinase levels below the lowest standard (2.0 to 30.1 ng mL^–1). Oxytocin-treated mares had median serum oxytocinase levels from 39 to 61 ng mL^–1 on Days 12 to 15. Early pregnant mares had detectable levels from Day 8 to 21 (medians ranging from 40 to 89 ng mL^–1). We concluded that serum oxytocinase levels were below the lowest standard in diestrus, and were low but detectable in oxytocin-treated mares. The highest oxytocinase levels were measured during early pregnancy. Further studies of serum oxytocinase in a larger population of mares, along with studies of tissue mRNA levels of oxytocinase, are required to better understand the regulation of oxytocin in horses.

Research was supported by the Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada) and the Equine Health Research Fund (University of Saskatchewan, SK, Canada).