Immunocontraceptive Efficacy of Native Porcine Zona Pellucida (pZP) Treatment of Nevada's Virginia Range Free-Roaming Horse Population.

In North America, range constraints due to burgeoning development increasingly encroach on wild horse habitat and necessitate effective but humane reproductive management. The largest free-roaming wild horse fertility control program by population (>3500) and territory size (≈300,000 acres) is located within Nevada's Virginia Range. Data from a field study investigated porcine zona pellucida (pZP) immunocontraception via remote dart delivery to mares in this population. Analyses aimed to measure efficacy by treatment effects on annual birth rates and population demographics and to evaluate treatment frequency and season against these variables. Analyses included mares' monthly data (January 2019-December 2022; 48 months), characterized by cumulative vaccination numbers subset into four classifications considering the vaccine as having no loss of efficacy or a loss within a 6-, 12-, and 18-month period post vaccination; from foaling data, the likelihood of being in foal and of conceiving in that month; and from age, as mature or immature (<1 years-old). A downward foaling rate and trend in the numbers of mature mares, descriptively presented at monthly intervals, showed markedly declining annual seasonal breeding peaks, with no observed change in foaling season or duration. Within four years, population coverage surpassed 70% and was associated with a 58% reduction in foaling, with only a 10% conception rate. Vaccinated mares increased proportionally: assuming a 12-month decay rate, the system reached stability at an average ≈1.0 vaccination/mare/year, providing a robust recommendation for treatment frequency contributing to best management practices.

Efficacy and Safety of Native and Recombinant Zona Pellucida Immunocontraceptive Vaccines Formulated with Non-Freund's Adjuvants in Donkeys.

This study aimed to test zona pellucida (ZP) vaccines' immunocontraceptive efficacy and safety when formulated with non-Freund's adjuvant (6% Pet Gel A and 500 Μg Poly(I:C)). Twenty-four jennies were randomly assigned to three treatment groups: reZP (n = 7) received three doses of recombinant ZP vaccine; pZP (n = 9) received two doses of native porcine ZP; and Control group (n = 8) received two injections of placebo. Jennies were monitored weekly via transrectal ultrasonography and blood sampling for serum progesterone profiles and anti-pZP antibody titres. In addition, adverse effects were inspected after vaccination. Thirty-five days after the last treatment, jacks were introduced to each group and rotated every 28 days. Vaccination with both pZP and reZP was associated with ovarian shutdown in 44% (4/9) and 71% (4/7) of jennies, 118 ± 33 and 91 ± 20 days after vaccination, respectively (p > 0.05). Vaccination delayed the chances of a jenny becoming pregnant (p = 0.0005; Control, 78 ± 31 days; pZP, 218 ± 69 days; reZP, 244 ± 104 days). Anti-pZP antibody titres were elevated in all vaccinated jennies compared to Control jennies (p < 0.05). In addition, only mild local injection site reactions were observed in the jennies after treatment. In conclusion, ZP vaccines formulated with non-Freund's adjuvant effectively controlled reproduction in jennies with only minor localised side effects.

Effects of an anti-gonadoliberin releasing hormone vaccine on testicular, epididymal and spermatogenic development in the horse.

The effects of the GnRH vaccine Improvac® on testicular and epididymal morphometrics, histology and spermatogenesis were measured in 19 young (15-20 months) colts randomly assigned to one control (saline, castration at 57 days, n = 6) or either of two GnRH vaccine-treatment groups, T-57 (castration at 57 days, n = 7) or T-100 (castration at 100 days, n = 6), respectively. All were immunized on Day 0 with a single booster on Day 28. Excised testes and epididymides were weighed and processed for histology to measure tubule, epithelial and muscle dimensions, the ratio of interstitial tissue to seminiferous tubules and determine the stage of spermatogenesis. Testis volume, unchanged within controls, decreased in T-57 and T-100 groups by 50% and 70%, respectively. Treated colts' testes were significantly lighter than controls (64% relative difference); however, epididymal mass showed no significant differences between groups. Proportionally less seminiferous tubule relative to interstitial tissue was observed in both treatment groups (5%) versus controls (22%) with a mean tubule size 28% smaller than controls. Controls exhibited a high proportion of seminiferous tubules with advanced stages of spermatogenesis, whereas treated colts showed a high proportion of tubules in the early stages of spermatogenesis. In conclusion, immunization against GnRH in prepubertal colts was effective at reducing the development of their intra-scrotal reproductive organs and preventing normal spermatogenesis. GnRH vaccination of young colts effectively and consistently reduced testis mass, tubule size and relative proportion of seminiferous tubule tissue while retarding spermatogenesis. The epididymis showed changes with a smaller tubule diameter, lower epithelial height and thicker muscle layer recorded in treated compared to control colts.

Unilateral salpingectomy in Sprague Dawley rats and its effect on litter size.

The study described a novel, rapidly performed, successful and safe surgical procedure in rats to achieve a reduction in the number of conceptuses. The objectives were to investigate the total foetal count and foetal health in both uterine horns after unilateral salpingectomy compared to the control group. Sixteen female Sprague-Dawley rats (Rattus norvegicus) were allocated to the study of which 10 rats underwent unilateral salpingectomy with six controls before all 16 were mated at 8-10 weeks of age. Gestational length was taken as 22 days coma? determined from the day of appearance of the copulatory plug. The female rats were terminated on day 19 or 20 of the gestational period. The foetuses in each horn were mapped and counted for comparison between the salpingectomy and control groups. The gravid uteri, individual foetal weights and placental weights were measured and compared between the two groups. This study described a novel, rapidly performed, successful and safe surgical procedure in rats. The mean number of foetuses in the salpingectomy group was significantly smaller than the control group. No significant differences in foetal and placental development were observed between the groups. These observations support future investigation of unilateral salpingectomy in other species as an alternative surgical method for population control.

Vaccination against GnRH as a prelude to surgical castration of horses.

BACKGROUND: Surgical castration is one of the most frequently performed surgeries in horses and is associated with various post-surgical complications ranging in severity. OBJECTIVES: We investigated the effects of administering an anti-GnRH vaccine to colts for reduction of testis size on the incidence of complications associated with their subsequent surgical castration. STUDY DESIGN: Randomised open controlled trial. METHODS: Nineteen colts were randomly assigned to one of the three groups. Treatment Groups V1-57 and V2-100 received two treatments of Improvac® (Zoetis, South Africa) before their castration in the field on either Day 57 or Day 100 following first treatment, respectively. Controls similarly received placebo treatments followed by castration on Day 57. Serial measurements of testicular dimensions and serum samples for measuring serum testosterone concentrations (STCs) and anti-GnRH antibody titres (ABTs) were obtained pre- and post-vaccination and on the day of castration. Clinical data recorded for 10 days determined post-surgical complications. RESULTS: All vaccinated colts showed a baseline STC concentration at castration with a strong ABT response. Mean testicular volume of Groups V1-57 and V2-100 reduced by 49.7% and 30.8%, respectively, on Day 57 and this further reduced in Group V2-100 by 63.9% at castration on Day 100. Testis size was significantly correlated with post-surgical preputial (P = 0.001) and scrotal (P = 0.025) swelling. MAIN LIMITATIONS: A study population of young light horse breed colts and a relatively small sample size limited this pilot study. CONCLUSIONS: Administering two doses of Improvac® in colts 28 days apart prior to castration effectively reduced their testicular volumes with associated significant post-surgical improvement in both the incidence and degree of local swelling. These results may inform mitigation of potential post-surgical complications associated with castration in the field.

Distribution of uterine histological changes in aged captive cheetahs (Acinonyx jubatus).

The histological effect on the felid uterus of sterilization, via ovariectomy or salpingectomy, is currently unknown. To investigate the association of ovariectomy or salpingectomy with uterine health, it is first necessary to establish if changes are distributed evenly throughout the uterus. Both laparoscopic ovariectomy and salpingectomy with concurrent sampling of the tip of the uterine horn are possible in the cheetah. Currently accepted practice for histopathological screening of the uterus utilizes four biopsy samples. It is not known whether this method accurately reflects the status of the entire uterus. In this study we histologically examined the uteri of six older cheetahs (one 7-year-old and five 10-10.5-year-old animals) via 21 tissue samples (three samples from seven different anatomical regions) per cheetah to determine overall uterine health. Although no defined lesions were detected, mild endometrial gland dilation, assumed to be of no functional consequence, was observed in multiple samples. The odds of observing this dilation was lowest in the uterine body and progressively increased in a cranial direction, being significantly higher at the tip of the uterine horns (OR = 11.5; 95% CI, 2.0-65.1; p = 0.006). This supported the reliability of sampling the tip of the uterine horn to screen for endometrial gland dilation.

Efficacy and safety of native and recombinant zona pellucida immunocontraceptive vaccines in donkeys.

Feral and semi-feral donkeys are recognised as a problem in some world regions. The main problem associated with uncontrolled donkey populations is habitat degradation and competition for feed resources, especially in arid climes. Controlling population numbers would reduce the impact of donkeys and other species. While removal by various means is effective, it has been shown to stimulate reproductive rate. Probably the most effective and humane solution is reducing reproduction using minimally invasive methods including immunocontraception. This study tested the immunocontraceptive efficacy and safety of zona pellucida (ZP) vaccines, both recombinant (reZP; three treatments) and native porcine (pZP; two treatments) vaccines formulated with Freund's modified complete (primary) and Freund's incomplete (boosters) adjuvants in donkey jennies. Control jennies received adjuvants only (two treatments). Twenty-five non-pregnant jennies were randomly assigned to reZP (n = 9), pZP (n = 8) or control (n = 8) groups. Weekly monitoring of the reproductive tract and ovaries via transrectal palpation and ultrasound and inspection of injection sites was conducted and anti-pZP antibody titers were measured. Five weeks after last treatment, one donkey jack was introduced to each group and rotated every 21 days. By 232 days after last treatment the number pregnant and median days to pregnancy was 2/9 and 214 (reZP group), 1/8 and 196 (pZP group) and 8/8 and 77 (control group). Median time to ovarian shut-down was 77 (9/9) and 56 (7/8) days for reZP and pZP groups, respectively. This was observed in association with a distinct reduction in mean uterine diameter. The antibody response was equally good for both ZP-treated groups. Incorporation of Freund's adjuvants initially produced a high incidence of side effects from local swelling and intermittent lameness followed weeks later by sterile abscesses (reZP, 9/9; pZP, 7/8; control, 3/8). Both ZP vaccines effectively controlled reproduction in jennies, albeit with a high incidence of adjuvant-associated side effects.

Direct culture-independent sequence typing of Taylorella equigenitalis obtained from genital swabs and frozen semen samples from South African horses.

We report herein the use of crude extracts obtained from samples of Taylorella equigenitalis-infected horses for the purpose of multi-locus sequence typing (MLST). Samples (n = 36) were collected from horses in South Africa from 1996 to 2017: 34 from genital swabs (stored at -20°C for 2-3 y) and 2 from cryopreserved raw semen aliquots (stored at -70°C for 18 y) prior to assay. The MLST assay showed a single sequence type (ST), designated ST4, that supported a point introduction and thus a common source for the South African outbreak of contagious equine metritis.

Researching immunocontraceptive vaccines with mares (Equus caballus) as both a target and model for African elephant (Loxodonta africana) cows: A review.

A sequence of studies is reviewed that reported the domestic horse (Equus caballus) mare as an appropriate and accessible research platform for recording clinical and laboratory data post-immunisation with anti- GnRH and -zona pellucida (ZP) immunocontraceptive vaccines. Experience with a native porcine ZP (pZP) vaccine in African elephant (Loxodonta africana) cows highlighted needs for improving vaccine formulations and more clearly defining associated ovarian effects and safety profiles. Initially, the efficacy, reversibility and safety of the GnRH vaccine Improvac® in mares was demonstrated using reproductive tract ultrasonography and concurrently measuring serum antibody titres and progesterone concentrations. Results informed the study design and minimally invasive monitoring of post-treatment ovarian steroid responses of this vaccine in free-ranging African elephant cows. A subsequent sequence of studies reported reversible contraceptive and immunological efficacy in pony mares immunised with pZP formulated with Freund's adjuvants. By comparison, mares treated with a recombinant ZP3 and ZP4 (reZP) vaccine showed disappointing responses. Unexpectedly, most pZP-treated mares showed ovarian inactivity. In attempting to understand this response, results showed the involvement of cytotoxic (CD8+) T-cells negatively correlated to serum ovarian steroid and anti-Müllerian hormone (AMH) levels. Of concern was the prevalence of injection-site lesions ascribable to Freund's adjuvants. Following this, mares treated with both pZP and a novel reZP vaccine formulated with non-Freund's adjuvants showed comparable immunological responses and ovarian inactivity, notably without adverse treatment reactions. In addition, measuring AMH showed promise for monitoring ovarian function in anti-ZP-treated animals.

Failure to detect equid herpesvirus types 1 and 4 DNA in placentae and healthy new-born Thoroughbred foals.

Equid herpesvirus type 1 is primarily a respiratory tract virus associated with poor athletic performance that can also cause late gestation abortion, neonatal foal death and encephalomyelopathy. Horizontal transmission is well described, whereas evidence of vertical transmission of equid herpesvirus type 1 associated with the birth of a healthy foal has not been demonstrated. This study sampled a population of Thoroughbred mares (n = 71), and their healthy neonatal foals and foetal membranes, to test for the presence of both equid herpesvirus types 1 and 4 using a quantitative polymerase chain reaction assay. Foetal membrane swabs and tissue samples were taken immediately post-partum, and venous blood samples and nasal swabs were obtained from both mare and foal 8 h after birth. Neither equid herpesvirus type 1 nor equid herpesvirus type 4 nucleic acid was detected in any sample, and it was concluded that there was no active shedding of equid herpesvirus types 1 and 4 at the time of sampling. Consequently, no evidence of vertical transmission of these viruses could be found on this stud farm during the sampling period.

Serum antibody immunoreactivity and safety of native porcine and recombinant zona pellucida vaccines formulated with a non-Freund's adjuvant in horses.

Commercial and regulatory limitations associated with native porcine zona pellucida (pZP) vaccines formulated with Freund's adjuvants may be overcome by developing effective recombinant ZP vaccines (reZP) and identifying alternative adjuvant formulations. A two-part preparatory study used 15 geldings and identified potentially effective alternative adjuvant formulations based on anti-pZP antibody response following treatment with pZP formulated with Addavax (AddaVax ™, Invivogen), Quil A (Quil-A® Adjuvant, Invivogen), Quil A and Poly (I:C) (HMW VacciGrade™, Invivogen), Pet Gel A (Montanide™ Pet Gel A, Seppic) and Pet Gel A and Poly (I:C). Injection site reactions, rectal temperature and respiratory and heart rates were also monitored for three days post-treatment. Suitable anti-pZP antibody titres were seen in response to Pet Gel A and Pet Gel A and Poly (I:C). Subsequently in 31 mares, following administration of pZP, reZP and a combination of pZP and reZP proteins prepared in Pet Gel A and Poly (I:C), both serum anti-pZP and -reZP antibody responses were monitored. In addition, safety was assessed for up to seven days post-treatment by inspection and palpation of gluteal intramuscular injection sites and measurement of rectal temperature. The measured antibody titres in all treatment groups differed significantly to an adjuvant control group (P < 0.001). Temporal changes in both anti-pZP and -reZP antibody titres in all ZP treatment groups were similar to patterns reported previously in various species vaccinated with pZP formulated with Freund's adjuvants. There were no differences in anti-pZP antibody titres between the pZP and reZP treated groups (P > 0.05). Side effects were mild and transient in nature. This represents the first application of a reZP vaccine formulated with non-Freund's adjuvants evoking a similar antibody titre response to native pZP vaccination in mares.

Ovarian function following immunocontraceptive vaccination of mares using native porcine and recombinant zona pellucida vaccines formulated with a non-Freund's adjuvant and anti-GnRH vaccines.

An important determinant in the selection of any contraceptive agent is the impact on ovarian function, both in the short and longer term. In this study, ovarian activity was monitored in mares immunised with one of the following vaccine formulations; native porcine zona pellucida (pZP), recombinant zona pellucida proteins ZP3 and ZP4 (reZP), pZP and reZP combined or a commercially available anti-GnRH vaccine. The ZP antigens were prepared in an adjuvant formulation consisting of 6% polymeric adjuvant (Montanide™ PetGel A, Seppic, France) and 500 µg polyinosinic-polycytidylic acid - TLR3-agonist (Poly(I:C) HMW VacciGrade™, Invivogen, USA). A vehicle-only control group was administered the adjuvant formulation without antigen. Ovarian activity was monitored using clinical observations (transrectal palpation and ultrasonography of the reproductive tract) in addition to blood sampling for serum progesterone and anti-Müllerian hormone (AMH) concentrations while employing a low sampling frequency. Treatments and measurements were initiated in December (southern hemisphere summer) and subsequent data collection was performed in January, February, March and May. Both reZP and anti-GnRH vaccination were associated with clinically evident ovarian suppression in the short term. Ovarian activity in mares administered a reZP or anti-GnRH vaccine was significantly different to adjuvant control and pZP treated mares. Serum AMH concentrations were different between pZP and anti-GnRH treated mares 3.5 months after the final vaccination. Serum AMH concentrations were significantly correlated with mare age, serum progesterone and ovarian volume.

Ovarian dysfunction associated with zona pellucida-based immunocontraceptive vaccines.

Despite more than 40 years of research into zona pellucida (ZP)-based vaccines, relatively little is known about their mechanism of action. Early research demonstrated precipitation of ZP glycoproteins by antiovarian antiserum, rendering oocytes resistant to sperm binding in vitro. Subsequent work showed significantly decreased fertilization rates following passive immunization, sparking interest in anti-ZP immunocontraception for human and animal use. The primary mechanism of action of ZP vaccines is generally considered to be an antibody-mediated interference with sperm-oocyte binding and/or fertilization. However, this mechanism of action excludes the potential for ovarian dysfunction associated with anti-ZP treatment in some species. A review of relevant literature in pertinent model, domestic and wildlife species reveals a variety of previous and current hypotheses for ovarian effects following ZP-based immunization. Ovarian dysfunction has been suggested to be a species-specific response. In addition, cytotoxic T-lymphocytes and the use of Freund's adjuvants have been suggested to play a role. Finally, the type and extent of glycosylation of ZP antigens have been proposed to influence ovarian effects. The validity of these hypotheses is re-examined in the light of current knowledge. Further investigation of ovarian function in species believed to be resistant to the ovarian effects of anti-ZP vaccines is warranted. To this end, anti-Müllerian hormone may provide a novel tool for the assessment of ovarian function during ZP-based immunocontraception, particularly in wildlife species not amenable to frequent clinical examination.

Missense Mutation in the Ligand-Binding Domain of the Horse Androgen Receptor Gene in a Thoroughbred Family with Inherited 64,XY (SRY+) Disorder of Sex Development.

Disorders of sex development (DSD) have long been documented in domestic animal species including horses. However, there is only a single report of an androgen receptor (AR) mutation causative of such a DSD syndrome in a horse pedigree. Here, we present a new familial AR mutation in horses. A missense mutation (c.2042G>C) at AR exon 4 explains the segregation of the DSD in a Thoroughbred horse pedigree. The mutation, expected to affect the ligand-binding domain of the AR protein, led to complete androgen insensitivity of 64,XY SRY+, testicular DSD individuals. Additionally, the design of a PCR-RFLP technique provided an accurate molecular test for the identification of horses carrying the mutation.

Draft Genome Sequence of Taylorella equigenitalis Strain ERC_G2224 Isolated from the Semen of a Lipizzaner Stallion in South Africa.

Taylorella equigenitalis is the causative agent of contagious equine metritis (CEM), a sexually transmitted infection of horses. We report here the genome sequence of T. equigenitalis strain ERC_G2224, isolated in 2015 from a semen sample collected in 1996 from a Lipizzaner stallion in South Africa.

Single-Incision Laparoscopic Sterilization of the Cheetah (Acinonyx jubatus).

OBJECTIVE: To describe laparoscopic ovariectomy and salpingectomy in the cheetah (Acinonyx jubatus) using single-incision laparoscopic surgery (SILS). STUDY DESIGN: Prospective cohort. ANIMALS: Female cheetahs (Acinonyx jubatus) (n = 21). METHODS: Cheetahs were randomly divided to receive either ovariectomy (n = 11) or salpingectomy (n = 10). The use and complications of a SILS port was evaluated in all of cheetahs. Surgery duration and insufflation volumes of carbon dioxide (CO2 ) were recorded and compared across procedures. RESULTS: Laparoscopic ovariectomy and salpingectomy were performed without complications using a SILS port. The poorly-developed mesosalpinx and ovarian bursa facilitated access to the uterine tube for salpingectomy in the cheetah. The median surgery duration for ovariectomy was 24 minutes (interquartile range 3) and for salpingectomy was 19.5 minutes (interquartile range 3) (P = .005). The median volume of CO2 used for ovariectomy was 11.25 L (interquartile range 3.08) and for salpingectomy was 4.90 L (interquartile range 2.52), (P = .001) CONCLUSIONS: Laparoscopic ovariectomy and salpingectomy can be performed in the cheetah using SILS without perioperative complications. Salpingectomy is faster than ovariectomy and requires less total CO2 for insufflation.

Detection of equine herpesvirus-4 and physiological stress patterns in young Thoroughbreds consigned to a South African auction sale.

BACKGROUND: The prevalence of equine herpesvirus types-1 and -4 (EHV-1 and -4) in South African Thoroughbreds at auction sales is currently undefined. Commingling of young Thoroughbreds from various populations together with physiological stress related to their transport and confinement at a sales complex, may be associated with shedding and transmission of EHV-1 and -4. This prospective cohort study sampled 90 young Thoroughbreds consigned from eight farms, originating from three provinces representative of the South African Thoroughbred breeding demographic to a sales complex. Nasal swabs for quantitative real-time polymerase chain reaction (qPCR) assay to detect EHV-1 and -4 nucleic acid and blood samples for enzyme-linked immunosorbent assay for EHV-1 and -4 antibodies were collected from all horses on arrival and departure. Additional nasal swabs for qPCR were obtained serially from those displaying pyrexia and, or nasal discharge. Daily faecal samples were used for determination of faecal glucocorticoid metabolite (FGM) concentrations as a measurement of physiological stress and these values were modelled to determine the factors best explaining FGM variability. RESULTS: EHV-4 nucleic acid was detected in 14.4 % and EHV-1 from none of the animals in the study population. Most (93.3 %) and very few (1.1 %) of this population showed antibodies indicating prior exposure to EHV-4 and EHV-1 respectively. Pyrexia and nasal discharge were poor predictors for detecting EHV-4 nucleic acid. The horses' FGM concentrations increased following arrival before decreasing for most of the remaining study period including the auction process. Model averaging showed that variation in FGM concentrations was best explained by days post-arrival and transport duration. CONCLUSIONS: In this study population, sales consignment was associated with limited detection of EHV-4 nucleic acid in nasal secretions, with most showing prior exposure to EHV-4 and very few to EHV-1. The physiological stress response shown by most reflected the combination of stressors associated with transport and arrival and these are key areas for future investigation into management practices to enhance health and welfare of young Thoroughbreds during sales consignment.

Reflections on psychoanalytic treatment of Lubavitch Chassidim couples: working with a culturally divergent population.

Chassidic Jews create separate developmental lines for males and females beginning at three years of age. Since early marriages are encouraged and there is minimal contact between the sexes prior to marriage, problems inevitably arise in relationships. This article discusses both newlywed and long-term married Lubavitch Chassidim in couples treatment with secular analysts, parameters necessary for successful treatment, and countertransferences that arise. It is part of an ongoing series of publications based on the authors' decade-long psychoanalytic work with this population.

The effect of consignment to broodmare sales on physiological stress measured by faecal glucocorticoid metabolites in pregnant Thoroughbred mares.

BACKGROUND: Validation of a method for the minimally-invasive measurement of physiological stress will help understanding of risk factors that may contribute to stress-associated events including recrudescence of Equid herpesvirus (EHV), which is anecdotally associated with sales consignment of pregnant Thoroughbred mares. In this study we compared two similar groups of late-gestation Thoroughbred broodmares on the same farm: a consigned Sales group (N = 8) and a non-consigned Control group (N = 6). The Sales mares were separated from their paddock companions and grouped prior to their preparation for, transport to, and return from the sales venue. Both groups were monitored by sampling at regular intervals from 5 days prior to until 14 days after the sales date (D0) to measure physiological stress in terms of changes in faecal glucocorticoid metabolite (FGM) concentrations, and for event-related viral recrudescence via daily body temperature measurements and periodic nasal swabs for PCR analysis for EHV-1 and -4 DNA. RESULTS: In both groups, FGM levels increased post-sales before returning to pre-sales levels. Specifically, FGM concentrations in the Sales mares were significantly higher on D + 3 and D + 10 than on D-4 and D-3 (F = 12.03, P < 0.0001, Post hoc: P = 0.0003-0.0008) and in the Control group FGM concentrations were higher on D + 10 than D-4 (F = 5.52, P = 0.004, Post hoc: P = 0.005). Interestingly, mean FGM levels in Control mares were significantly higher at 4 of the 5 sampling points (t = 5.64-2.25, p = 0.0001-0.044). Only one (Sales) mare showed PCR evidence of EHV-1 shedding. CONCLUSIONS: Using FGM to measure physiological stress was supported by the increases observed in all mares after Sales consignment, including those not consigned to the sale. Monitoring FGM levels therefore represents an appropriate, minimally-invasive method for future studies to assess the contribution of physiological stress to EHV recrudescence in horses transported to sales or equestrian events.