Predictors of foaling outcomes in barren and maiden Thoroughbred mares in South Africa.

Population demographics and reproductive performance of Thoroughbred populations have been described, but the most recent assessment of the South African Thoroughbred population was reported two decades ago. Objectives of this study were to report demographic data for selected Thoroughbred breeding populations and to analyse selected mare-level variables in association with foaling outcomes, as predictors of reproductive performance. The National Horseracing Authority of Southern Africa's stud health scheme requires annual screening of Thoroughbred stallions, maiden and barren mares for venereal pathogens prior to breeding. In 2018 and 2019, 1 065 and 1 207 horses were sampled, respectively. Demographic data were sourced from laboratory sample submission forms that accompanied samples and supplemented by data gathered from the annual Thoroughbred foal identification programme. Univariate analysis of candidate predictors of successful foaling outcomes was performed followed by assessment in a multivariable model.Median ages of mares and stallions tested in 2018 and 2019 were nine and 11 years, respectively. Nearly twice the number of barren compared to maiden mares were tested in each year, and failure to conceive was the most common reported reason for classification as barren. Of mares tested in 2018 and 2019, 68.1% (95% CI 65.1, 70.9) and 63.3% (95% CI 60.4, 66.1), respectively, subsequently produced foals that were presented for identification. Mare age, rather than reproductive status, was a significant predictor of having a foal presented for identification. In conclusion, novel demographic data were described for South African Thoroughbred populations. Seasonal foaling rate as the selected measure of reproductive performance for sampled mares ranged from 63.3% to 68.1% and declined with increasing mare age.

Ultrasonographic evaluation of fetal development in the rat.

The study objectives were to measure gestational sac (GS) diameter and crown-to-rump (CR) length in conscious pregnant rats and to determine the chronological ultrasonographic appearance of heart beat and fetal organogenesis. The study formed part of a unilateral surgical salpingectomy trial with 16 female Sprague-Dawley rats (Rattus norvegicus). Ten rats were operated on while the other six served as controls. After surgery all were mated at 8-10 weeks of age. Gestational length was taken as 22 days. Rats were manually restrained for abdominal ultrasonography and were scanned daily from day 7 until day 19 or 20 post-mating followed by immediate euthanasia. Measurements' were taken from two GSs per rat. The presence of several early GSs in a row were described as a "string of pearls". As a fetus developed features of organogenesis were recorded. There was no significant difference in GS diameters and CR length between the test and control groups. Mean GS diameters ranged from 2.9 mm (day 7) to 18.1 mm (day 20). A string of pearls was seen on days 8-11. The CR length ranged from 1.6 mm (day 11) to 26.4 mm (day 20). A heart beat was first seen on day 11 and the echogenic vertebral column at day 14-15. From day 16, ribs, feet and the isoechoic lungs and liver were seen. The lungs became hyperechoic to the liver from day 18-19. The tail and mandible were visible on day 18 and the aorta and caudal vena cava were seen on day 19, as well as an occasional bladder. These measurements and staged in utero ultrasonographic appearance of various organ and skeletal structures will assist in a reasonably accurate prediction of the day of impending parturition by laboratory personnel and researchers.

Antigen-specific CD4+ and CD8+ T-cell responses in PBMC from pony mares immunized with either native or recombinant zona pellucida vaccines.

Few studies have investigated the cell mediated immune response during zona pellucida-based immunocontraception, despite hypothesized cytotoxic T-cell involvement in ovarian dysfunction associated with these vaccines. This study aimed to investigate antigen-specific anamnestic responses of helper (CD4+) and cytotoxic (CD8+) T-lymphocytes in peripheral blood mononuclear cells (PBMC) isolated from pony mares before and after their treatment with native porcine zona pellucida (pZP), recombinant pZP3 and pZP4 antigens (reZP) or adjuvanted saline. Mares were randomly assigned to pZP, reZP and control groups (n = 7 per group). Treatments consisted of a primary vaccination or saline (V1; Day 0) incorporating Freund's modified complete adjuvant, followed by a single booster (V2; Day 35) incorporating Freund's incomplete adjuvant. Peripheral blood mononuclear cells were isolated and cryopreserved immediately prior to V1 (Day 0) and five weeks post V2 (Day 70). Relative proliferation of T-lymphocytes in response to pZP antigen was assessed using carboxyfluorescein diacetate succinimidyl ester dilution with immunophenotyping, analysed via flow cytometry. Significant pZP-specific CD4+ and CD8+ T-lymphocyte responses were detected in PBMC isolated from mares treated with either pZP or reZP, in comparison to pre-treatment samples. In the pZP group, but not the reZP group, CD8+ T-cell proliferation showed significant negative correlations to circulating progesterone, oestradiol and anti-Müllerian hormone levels. Results suggest that antigen-specific CD8+ T-cells may play a role in ovarian suppression observed during pZP immunocontraception in this species.

Influence of cycle stage, age and endometrial biopsy score on oxytocin receptor distribution and gene expression in the cervix and uterus of non-pregnant mares.

Persistent breeding-induced endometritis (PBIE) or delayed uterine clearance (DUC) are major causes of mare subfertility. Oxytocin and its receptor are thought to play significant roles in the pathogenesis of DUC but the specific roles of oxytocin receptor (OR) distribution and gene expression remain undefined. In this study both OR distribution and gene expression in the endometrium, myometrium and cervix during both luteal and non-luteal phases in non-pregnant mares (n = 27) of differing age (young: 2-9 years, n = 17; old: > 10 years, n = 10) and endometrial biopsy score were described using immunohistochemistry (IHC) and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Immunohistochemistry showed a similar pattern of OR distribution in uterus and cervix, with the exception of the glandular epithelium, absent in the cervix. Uterine ORs were localized in endometrial luminal and glandular epithelia, transmural vascular endothelium, sub-epithelial and peri-glandular stromal cells and myometrial smooth muscle cells. The OR labeling intensity was consistently greatest in the vascular endothelium. Real-time qPCR showed a higher OR gene expression in myometrium compared to cervix (P = 0.001) and endometrium (P = 0.009). There was no difference in OR gene expression between cervix and endometrium (P = 1.0). Oxytocin receptor gene expression was significantly higher during the non-luteal phase in both combined uterine tissues (endometrium and myometrium) and myometrium. Oxytocin receptor distribution and gene expression were not influenced by a mare's age or endometrial biopsy score. As endometrial biopsy score and mare age were not predictors of OR gene expression, deficient OR gene expression is unlikely to be associated with DUC.

Serum anti-Müllerian hormone dynamics in mares following immunocontraception with anti-zona pellucida or -GnRH vaccines.

Circulating anti-Müllerian hormone concentration (AMH) is positively correlated to the number of small growing follicles in the mare and may reflect ovarian function. Dynamics of AMH during immunocontraception have not previously been investigated. This study aimed to compare serum AMH in mares following treatment with native porcine zona pellucida (pZP), recombinant pZP3 and pZP4 (reZP) or gonadotrophin releasing hormone (GnRH) vaccines, and saline-treated controls. Stored sera collected during two previous studies examining ovarian activity in mares during zona pellucida (ZP) or GnRH immunocontraception were analysed for serum AMH. Data were compared among treatment groups using mixed-effects linear regression and one-way ANOVA with post hoc testing. Correlations between AMH and previously reported clinical variables were estimated using Spearman's rho. Mares immunized against GnRH showed variable but detectable AMH throughout successive breeding and non-breeding seasons that were not significantly different to unvaccinated control mares. Mares treated with pZP demonstrated marked, reversible suppression of AMH. Mares immunized using reZP showed an intermediate effect. In the ZP study, AMH was positively correlated to serum progesterone concentrations, mean ovarian volumes and antral follicle counts, whereas no correlations between AMH and serum progesterone concentrations, mean ovarian volumes, or the presence of one or more follicles ≥20 mm in diameter were detected in the GnRH study. In conclusion, marked suppression of AMH during pZP immunocontraception, but not during GnRH immunocontraception, suggested enhanced suppression of ovarian follicular development and, or follicular function during pZP immunocontraception. Serum AMH concentrations may provide a novel tool for the assessment of ovarian function during ZP-based immunocontraception.

Ovarian function and pregnancy outcome in pony mares following immunocontraception with native and recombinant porcine zona pellucida vaccines.

REASONS FOR PERFORMING STUDY: Few studies have investigated ovarian function in the mare undergoing porcine zona pellucida (pZP) immunocontraception despite reported ovarian dysfunction in other species. OBJECTIVES: This study aimed to describe ovarian function and oestrous cyclicity in pony mares following treatment with either the conventional pZP vaccine or a novel recombinant form of the vaccine derived from porcine ZP3 and ZP4 (reZP). In addition, the contraceptive efficacy of pZP vs. reZP was assessed. STUDY DESIGN: Blinded, randomised, prospective clinical trial. METHODS: Mares (n = 21) were randomised into 3 groups of 7: Group I received the pZP vaccine, with a booster 5 weeks later; Group II received the reZP vaccine, with a booster 5 weeks later; and Group III (controls) received 2 treatments, 5 weeks apart, of saline and adjuvant alone. Mares underwent weekly monitoring via transrectal palpation and ultrasound examination of the reproductive tract, with daily monitoring during oestrus. Data were collected over a 24 week period coinciding with the physiological breeding season; treatments commenced in Week 4. Serum samples were obtained for antibody titres and ovarian steroid level analyses at 7 day intervals. Cycling mares were bred via fresh semen artificial inseminations over a maximum of 2 consecutive oestrous cycles, commencing 5 weeks post booster vaccination. RESULTS: Control mares cycled throughout the trial. After treatment, 6 of 7 pZP mares (86%) and one reZP mare (14%) had an extended anoestrus that correlated with basal serum oestradiol and progesterone levels. All mares resumed cyclicity by 10 months post treatment. Pregnancies were diagnosed in all controls, 4 reZP- (57%) and none of the pZP-immunised mares. CONCLUSIONS: The current study demonstrates the reversible suppression of ovarian function in pony mares following treatment with pZP. The effect of the reZP vaccine on pregnancy outcome requires further investigation.

Polymerase chain reaction-based national surveillance programme to determine the distribution and prevalence of Taylorella equigenitalis in South African horses.

REASONS FOR PERFORMING STUDY: The response to the first outbreak of contagious equine metritis in South Africa included pioneering a web-based platform to coordinate key aspects of a national, real-time polymerase chain reaction (qPCR)-based stallion screening programme to determine the distribution and prevalence of Taylorella equigenitalis in stallions and exposed mares. OBJECTIVES: To define the hypothesised pre-existing status of T. equigenitalis in the South African equine population and progression of the epidemiological investigation via the implementation of a molecular diagnostic-based surveillance programme. STUDY DESIGN: Retrospective case series. METHODS: Screening for T. equigenitalis was via a qPCR assay on genital swabs obtained from predilection sites in stallions and mares with subsequent confirmation using bacterial culture according to prescribed methods. RESULTS: The initial outbreak investigation identified 4 horses including the index stallion and mare. Traceback of in-contact horses identified 26 horses, including a subpopulation focus at the South African Lipizzaner Centre where 24/33 resident stallions tested positive for T. equigenitalis on qPCR. The national screening programme identified an additional 9 stallions. A total of 39 horses (36 stallions and 3 mares) tested positive for T. equigenitalis by qPCR and T. equigenitalis was isolated from 23 of these stallions and 2 of these mares. In addition to the index property, an artificial breeding centre where the index case was first identified, an additional 12 properties with infected horses were identified in 3/9 provinces. Horses on 11 of these 12 properties were directly linked to the index property. Two incidents of T. equigenitalis transmission associated with artificial insemination were recorded. CONCLUSIONS: T. equigenitalis was present in a subpopulation focus within the South African horse population prior to the outbreak identification in April 2011. Horizontal fomite-associated spread was the most probable route of transmission between stallions. The targeted surveillance of stallions and exposed mares using a qPCR-based screening programme expedited investigation of the distribution and prevalence of T. equigenitalis infection in South African horses. The application of qPCR provided a sensitive and practical screening test for identification of T. equigenitalis-positive animals as part of an emergency response to the first identified cases of T. equigenitalis infection in South African horses.

Ultrasonographic and laparoscopic evaluation of the reproductive tract in older captive female cheetahs (Acinonyx jubatus).

The study uniquely described the clinical value of transabdominal ultrasonography for monitoring features characterizing the estrous cycle in female cheetahs (Acinonyx jubatus). The reproductive tracts of 21 female, nulliparous, and relatively aged (median: 11 and interquartile range: 9.25-14 years) captive cheetahs resident on two sites in Namibia were assessed by transabdominal ultrasound. Subsequently, the ovarian findings on ultrasound were compared with direct visualization while performing laparoscopic sterilization. A combination of these observations supported by concurrent sampling for vaginal cytology and serum progesterone concentrations defined the estrous status of individual animals. At one site, six cheetahs had been implanted with the GnRH agonist, deslorelin as a contraceptive at least once within the preceding 11 years. On ultrasound, 31 uterine horns and 35 ovaries with discernible structures on 28 (86%) were visualized in the 21 cheetahs. The uterine body was difficult to visualize because of its intrapelvic location. Eleven of 19 uteri (58%) visualized showed endometrial edema suggestive of estrogenization. The uteri of four cheetahs (19%) showed evidence of mild cystic endometrial hyperplasia. Paraovarian cysts were seen on ultrasound (n = 21) and laparoscopy (n = 26) in 16 (76.2%) and 18 (85.7%) cheetahs, respectively. Ovarian volumes obtained from ultrasonographically determined dimensions predicted cyclic activity. Laparoscopy showed that 19 ovaries had discernible follicular structures. In the study population, 10 (47.6%) cheetahs were in proestrus or estrus; none in the luteal phase; and 11 (52.4%) in anestrus. Transabdominal ultrasound, in combination with serum progesterone concentrations and vaginal cytology, was used with acceptable accuracy to assess cyclic ovarian activity in captive cheetahs. A considerable proportion of this aged population showed ovarian activity and the prevalence of paraovarian cysts was notable. A history of prior deslorelin treatment was not associated with either reproductive activity or uterine pathology.

Epidemiology and reproductive outcomes of EHV-1 abortion epizootics in unvaccinated Thoroughbred mares in South Africa.

REASONS FOR PERFORMING STUDY: Equine herpesvirus 1 (EHV-1) is one of the most common causes of infectious abortion in mares. Analysing the demography of outbreaks and detailing subsequent reproductive performance of affected mares will assist in the management of future (threatened) epizootics. OBJECTIVES: To examine the epidemiology and reproductive outcomes of 2 EHV-1 abortion epizootics with very different patterns of morbidity. STUDY DESIGN: Epidemiological and reproductive data were analysed retrospectively following abortion epizootics associated with EHV-1, but initiated via different routes, among unvaccinated mares on 2 Thoroughbred farms in South Africa. METHODS: Aborting mares were assigned to either the EHV-1 abortion cohort via positive immunostaining (Farms 1 and 2) or quantitative PCR (Farm 2) on tissue samples, or to the non-EHV abortion cohort. RESULTS: During their respective epizootics, EHV-1 abortions affected 9/30 (30.0%) and 18/316 (5.7%) of the pregnant mares on Farms 1 and 2, respectively; there were also 25 (7.9%) non-EHV abortions on Farm 2. Epizootic differences included: durations (Farm 1 = 135 days; Farm 2 = 34 days), intervals between first and subsequent abortions (Farm 1 = 39 days; Farm 2 = 2 days) and intervals to confirmation of EHV-1 (Farm 1 = 40 days; Farm 2 = 2 days). The median (range) age of EHV-1 abortion mares (8.0; 5-18 years) in both epizootics was similar but significantly younger (P = 0.004) than the 25 non-EHV-1 abortion mares (11.0; 4-24 years) on Farm 2. Gestation stage (median; range) of EHV-1 (291.5; 277-313) and non-EHV-1 (211.9; 61-339 days) abortions were significantly different (P = 0.001). The post abortion complications and subsequent reproductive outcomes had no significant association with EHV-1 abortion. CONCLUSIONS: The marked difference in morbidity between the 2 epizootics may be associated with routes of introduction or intervention strategy dictated by availability of molecular diagnostic techniques. Unexpectedly, EHV-1 was not more commonly associated with post abortion complications.

Reversibility of the effects of GnRH-vaccination used to suppress reproductive function in mares.

REASONS FOR PERFORMING STUDY: Active immunisation against gonadotrophin-releasing hormone (GnRH) provides a reversible method for control of oestrous behaviour and fertility in mares. Previous reports failed to demonstrate the interval to resumption of cyclic ovarian activity after GnRH-vaccination. HYPOTHESIS: Administration of the GnRH-vaccine Improvac in a large group of mares of various ages will result in effective, reliably reversible suppression of ovarian activity within a 2 year period. METHODS: The mares, subdivided into 3 age categories, were vaccinated twice (with a 35 day interval) using 400 µg Improvac and monitored via blood samples until Day 720 after initial vaccination for serum progesterone concentration determination by radioimmune assay and anti-GnRH antibody titre by enzyme immunoassay. Samples were collected until individuals resumed cyclic ovarian activity. RESULTS: All mares showed suppression of cyclic ovarian activity by clinical examination and serum progesterone concentration (SPC) ≤ 1 nmol/l by Day 70 and 92.2% resumed cyclic activity by SPC at Day 720 with a mean interval = 417.8 days (s.d. ± 23.9; range 232-488 days, median 344 days). A significant age effect (P = 0.028) on the interval, but not on GnRH-antibody titre response, was observed between the youngest (≤ 4 years) and oldest (≥ 11 years) categories. CONCLUSIONS: Immunising adult mares of all ages with Improvac resulted in a reversible suppression of cyclic ovarian activity in most mares. An age effect, with the youngest mares showing a longer interval to reversibility, was observed.

A predictive model for reproductive performance following abortion in Thoroughbred mares.

Pregnancy losses include early embryonic death (EED) and later (postimplantation) abortion. Abortions, particularly Equid herpesvirus (EHV-1) abortion epizootics, cause severe economic and production losses. The long-term effects of EHV-1 and other abortions on subsequent reproductive performance in broodmare populations, however, remain undefined. This study described the relationships of EED and abortion with the following reproductive outcomes in Thoroughbred systems: breeding efficiency, month of last breeding, subsequent pregnancy and live foal rates. A prospective cohort study in broodmare populations following EHV-1 epizootics on two South African farms was used to develop predictive models of the relative influences and interactions of reproductive variables associated with EHV-1 and other abortion causes on reproductive performance. EED predicted all the reproductive outcomes. Abortion predicted increased effort and month of breeding to establish pregnancy, but not becoming pregnant or foaling. Increasing age predicted decreased reproductive efficiency, and pregnancy and foaling probabilities. Mare reproductive status predicted breeding efficiency and the last month of breeding, but not establishing pregnancy. The last month of breeding predicted efficiency, pregnancy and foaling. Interestingly, breeding in the first month of the season was associated with an improved probability of pregnancy among barren mares.

The use of electrochemically activated saline as a uterine instillation in pony mares.

Twelve pony mares were randomly assigned to either a control or a treatment group and inseminated with fresh, raw semen from a single stallion of known fertility in a cross-over trial design. Pregnancy was diagnosed by transrectal ultrasound 12-14 days post-ovulation and then terminated by administration of a luteolytic dose of cloprostenol. Treatment mares received a uterine instillation of 100 ml of electrochemically activated (ECA) saline 4-12 hours post-insemination. Control mares received no treatment post-insemination. Per cycle pregnancy rate was 58.3 % in the control group and 50 % in the treatment group. There was no statistical difference (P = 1.000) in pregnancy rate between the 2 groups. The principles of ECA and applications of ECA saline are discussed.

A survey of reproductive success in South African Thoroughbred horse breeding from 1975 to 1999.

The data and an analysis of the statistical summaries from the 'Return of Mares' of the General Stud Book of The Jockey Club of Southern Africa from 1975-1999 are presented. The total number of mares covered per season ranged from 7393 (1992) to 5180 (1995). The total living produce in the period surveyed was 95 317 foals. The categories of data examined include: the total number of mares covered per season; the total numbers and percentage of their living produce; the total numbers and percentage dead produce, 'slips' and foals born dead, barren and 'no return' mares; and the total number of live twins reported. The percentage live foals per season increased from 52 to 62% and the percentage barren and 'no return' mares declined from 35.50 to 28.40% over the period surveyed. The number of live twins reported showed a dramatic reduction from 156 to 5. These apparent improvements are ascribed to a combination of factors including advances in veterinary knowledge and technology. The findings are similar to those reported by similar surveys of national Thoroughbred populations from North America and Germany. There is an indication to broaden this annual survey by recording additional parameters more accurately reflecting reproductive efficiency rather than a cumulative annual total of live foals.

Microsatellite analysis of cryopreserved stallion semen stored on FTA paper.

The aim of this study was to establish and validate a method to permit microsatellite analysis of DNA profiles obtained from frozen-thawed stallion sperm cells. This would provide reliable and accurate verification of the identification of a semen donor. Ejaculates from 5 pony stallions were collected, processed and frozen in 0.5 ml plastic straws. Aliquots of 100 microl of the frozen-thawed semen thus obtained were either placed directly, or diluted (1:10; 1:100; and 1:1000) and placed on slides of FTA paper. Similarly, blood samples obtained from each of the stallions were placed onto slides of FTA paper. A punch was removed from each sample after drying Each sample was mixed with FTA purification reagent, Dithiothreitol and Proteinase K before incubation and processing. All samples were processed with a set of 13 microsatellite markers. Further analysis permitted a comparison of the DNA profiles of the frozen-thawed semen and the blood samples. A full profile of markers was obtained from the 1:10 and 1:100 dilutions of the frozen-thawed semen samples as well as from the blood samples. The DNA profiles from the frozen-thawed semen and blood samples obtained from the stallions matched in all cases.

Use of the Accusport semi-automated analyser to determine blood lactate as an aid in the clinical assessment of horses with colic.

The most useful diagnostic methods in the initial evaluation of horses with colic assess the morphological and functional status of the gastrointestinal tract and cardiovascular status. This evaluation is best achieved using a combination of clinical and laboratory data. Blood lactate concentration (BL) is one of these variables. BL rises mainly due to poor tissue perfusion and anaerobic glycolysis associated with shock, providing an indicator of both the severity of disease and its prognosis. A hand-held lactate meter, Accusport, provides a rapid (60 seconds), inexpensive dry-chemical-based determination of BL. This trial evaluated the Accusport's ability to provide BL data as an adjunct to the initial clinical evaluation of horses with colic. The accuracy of the Accusport was tested by evaluation of its interchangeability with the benchmark enzymatic kit evaluation of BL in a trial using data collected firstly from 10 clinically normal control horses and subsequently from 48 horses presented with signs of colic. The BL values were recorded together with the clinical variables of heart rate (HR), capillary refill time (CRT), haematocrit (Hct), and pain character and severity on the initial assessment of the colic horses. Information regarding choice of therapeutic management (medical or surgical) and eventual case outcome (full recovery or died/euthanased) was recorded. The Accusport was found to be interchangeable with the enzymatic kit for recording BL values in colic horses with BL < 10 mmol/l, which is within the BL range associated with survival. The interchangeability of an additional, laboratory-based wet chemical assay for BL, the Stat 7 was simultaneously evaluated for the colic and control horses. The Stat 7 was found to be interchangeable with the enzymatic kit for BL determination of colic horses. No linear associations between BL values with HR, CRT, Hct or pain assessment were observed. No relationship with either selection of therapeutic method or eventual case outcome was observed. All horses with BL >8 mmol/l died or were euthanased.

Vulvovaginectomy and neo-urethrostomy for treatment of haemangiosarcoma of the vulva and vagina.

Vulvovaginectomy and neo-urethrostomy were performed in a 9-year-old German shepherd dog following a diagnosis of infiltrative vulvar and vestibulovaginal haemangiosarcoma. The dog was presented for intermittent vulvar haemorrhage over a 3-month period. On examination the vulva and vestibulovagina were distended and firm. Vaginal discharge and fine needle aspiration cytology detected anaplastic cells. Haemangiosarcoma was diagnosed on biopsy. A new urethral opening was created in the floor of the vagina allowing resection of the vulva and caudal vestibulovagina. Urinary continence was preserved and healing was without complications.

Datura seed intoxication in two horses.

A sunflower-based feed supplement grossly contaminated with the seed of a Datura sp. resulted in severe signs of poisoning in 2 horses. One horse died peracutely of acute gastric dilatation and rupture following ingestion of the contaminated feed. The 2nd horse developed unresponsive paralytic ileus that led to euthanasia. Examination of the feed and gastrointestinal contents of both horses showed a high proportion of the characteristic Datura sp. seeds. The clinical signs and pathology in both cases were consistent with intoxication by the parasympatholytic alkaloid components of Datura sp.