Call number: LD2668 .T4 1943 C6 / Master of Science

Master of Science / Department of Animal Sciences and Industry / David M. Grieger / Mares in estrus can exhibit erratic and fractious behavior that may adversely affect their ease of handling. Current treatments for suppression of estrus in the mare include an oral progestin, Regu-mate® (Hodgson et al., 2005), uterine insertion of a glass ball (Nie et al., 2003), fetal crushing through rectal palpation (Lefranc, 2004) and surgical sterilization (McCue, 2003). Although effective, all of these procedures are problematic for different reasons. Immunization against an endogenous hormone critical to fertility is an attractive alternative in suppressing estrus; especially if it is less intrusive and reversible. This strategy has been demonstrated by targeting gonadotropin-releasing hormone (GnRH) in gilts (Esbenshade and Britt, 1985), ewes (Clarke et al., 1978), mares (Garza et al., 1986), and heifers (Johnson et al., 1988). The antigen developed for this study was produced using the sequence of the bacterial protein, glutathione-S-transferase (GST) linked to three in-tandem repeats of the mammalian GnRH gene coding sequence (GST-GnRH3). Six reproductively sound mares, between the ages of 3 and 8 years, were used in this study. The anti-GnRH group (n=4) received 1 mg of GST-GnRH3 in 2ml of incomplete Freund’s adjuvant (IFA) as the primary injection. Four weeks later, mares received a single booster injection of 0.5mg of GST-GnRH3 in 1ml of IFA. The control mares (n=2) received similar doses of GST protein only, in identical injection volumes of IFA as the anti-GnRH group. The entire duration of the study ran for 22 weeks from early May through September. Ovaries were monitored three times weekly to track follicular growth and ovulation via transrectal ultrasonography. In addition, all mares were exposed to a stallion twice weekly and observed for estrous behavior. Weekly blood samples were collected to evaluate progesterone levels and serum binding of GnRH. The GST-GnRH3 antigen suppressed follicular activity in all treatment mares within 45 days following the second injection. Estrous behavior was suppressed in all but one mare in the anti-GnRH group. When exposed to the stallion, this mare displayed strong estrous behavior for seven weeks despite her lack of ovarian cyclicity. Follicular activity and estrous behavior remained normal in one of the control mares (avg. cycle length = 20 days). For the final 10 weeks of the study, however, the other control mare developed large follicles but failed to ovulate according to the ultrasound data. This mare did not display estrous behavior during this period, and her progesterone levels remained greater than 2 ng/ml for most of the final 10 weeks of the study. Approximately 2 weeks after the booster injection all anti-GnRH mares had progesterone levels of <1ng/ml. GnRH antibody binding peaked two weeks following booster immunization in all treated mares and remained undetectable in control mares throughout the study. The GST-GnRH3 treatment induced GnRH binding, suppressed follicular activity and reduced progesterone concentrations in all four mares. Although estrous behavior was abolished in 3 of the 4 treated mares, one did continue to demonstrate estrous behavior in the presence of a stallion. This dissociation of ovarian activity and estrous behavior was evident in our study with a limited number of animals, but the vaccine does show promise in reducing unwanted estrous behavior.

Research has centred on multidimensional chromatographic techniques which utilise the high specificity of immunoaffinity chromatography for extraction of analytes from complex biological matrices. On-line immunoaffinity chromatography-high performance liquid chromatography-mass spectrometry (IAC-HPLC-MS) systems (IAC and HPLC coupled via a loop interface) were developed for the confirmatory analysis of the corticosteroids dexamethasone and flumethasone with MS detection. Utilising an atmospheric pressure chemical ionisation (APCI) LC-MS interface, dexamethasone was confirmed in both spiked and post administration equine urine samples, with a detection limit of 0.1 ug 1-l. Detection by quadrupole ion trap mass spectrometry (ITMS) using a particle beam (PB) interface was performed for dexamethasone and flumethasone in post administration equine urine samples with high precision (6.9-7.4 %) with limits of detection in the range 3-4 ug 1-l. Studies were also conducted in this work into the antibody crossreactivity and non-specific binding of corticosteroids on a HEMA bound anti-dexamethasone lAC column. On-line IAC-HPLC and IAC-HPLC-GC have been developed and assessed for the determination of testosterone in equine urine. A novel approach to interfacing lAC with HPLC being achieved using a porous graphitic carbon (PGC) column. The IAC-HPLC system developed was used for sample pre-treatment for combustion isotope ratio mass spectrometry analysis. The IAC-HPLC and IAC-HPLC-GC systems finally being coupled with mass spectrometry to enable confirmation of the endogenous steroid at 0.5 ug 1-l and 1 ug 1-l respectively in stripped equine urine.

4 pp. / The Informed Arizona Equestrian Horse Health Series was designed to bring up to date information on issues of importance to the horse owners of Arizona and beyond. Rabies has been identified in horses in Arizona (most recently Santa Cruz county in 2016), and is not only fatal for horses, but also can affect the humans handling those horses. It is often overlooked as a possible diagnosis due to the nonspecific early signs of infection. Find out how to protect you and your horses from this devastating disease.

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Uterine inflammation is one of the causes of a poor uterine environment. This can result in early embryonic loss in the mare due to an inhibition of or an increased secretion of prostaglandin F2α (PGF2α ). Oxytocin binds to endometrial cell receptors to activate prostaglandin synthesis. Increased secretion or accumulation of PGF2α within the uterus due to uterine inflammation can cause luteolysis and result in early embryonic loss. Supplementation with polyunsaturated fatty acids (PUFAs) has been shown to influence prostaglandin production in many species, although the effects on the mare remain unknown. Equine endometrial biopsies were collected and used to establish endometrial epithelial cell and explant cultures to determine the release of PGF2α and PGFM in response to oxytocin stimulation. Endometrial explant cultures were used to determine the inhibitory effects of Atosiban, an oxytocin receptor antagonist, and Indomethacin, a cyclooxygenase –2 inhibitor, on PGF2α secretion. Endometrial explant cultures were challenged with oxytocin (250 nM) and PGF2α concentrations were measured over time. The effects of PUFAs on equine endometrial prostaglandin production were determined using endometrial biopsies harvested on day two of behavioral estrus. Equine endometrial cells were established and shown to replicate in culture and on a basement membrane matrix. Equine endometrial explants stimulated with oxytocin had increased secretion of PGF2α and PGE2 and the secretion of PGF2α was inhibited through an oxytocin receptor antagonist and Cox inhibition. Endometrial explants stimulated with lipopolysaccharide had increased secretion of PGF2α and PGE2, however oxytocin stimulated to a greater extent than LPS. Supplementation with PUFAs, specifically DHA, decreased the secretion of PGF2α and PGE2, however AA and EPA failed to influence this response. Expression of mRNA was not influenced by fatty acid supplementation, however was altered by stimulus. Therefore DHA influences the inflammatory response in vitro through mechanisms other than enzyme expression. Decreased PGF2α production associated with PUFA supplementation in vivo, creates a likely approach for decreasing early embryonic loss associated with post breeding inflammation commonly seen in the equine industry.

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