Evidence-based practice in canine artificial insemination.

A number of methods are currently used to predict the optimal date of insemination of the breeding bitch, particularly with the use of frozen-thawed canine semen which has a far shorter lifespan than fresh semen. Aside from confirming cytological oestrus, vaginal cytology is of no assistance in predicting the most fertile day(s) in a bitch; however, a neglected avenue of research suggests that vaginal cytology may be of great importance in confirming the days of optimal fertility retrospectively. Similarly, vaginoscopy provides clues as to the stage of a bitch's cycle but is inadequate as a sole determinant of her most fertile days. Nevertheless, vaginoscopy is useful to identify very late oestrus and the onset of dioestrus, as well as Stage I of labour (cervical dilatation). Due to variations in the rate at which circulating progesterone concentrations rise in individual bitches, the reliability of circulating progesterone concentrations for determining the optimal day(s) of insemination with frozen-thawed semen decreases as values rise. Moreover, progesterone assay results can vary widely due to extrinsic factors such as the time of blood sampling, sample storage conditions and the assay employed. Finally, this review investigates evidence surrounding various insemination routes and suggests that well-performed vaginal insemination, even with frozen-thawed semen, may be an acceptable approach for cases where transcervical insemination is impractical.

Sustained-release deslorelin acetate implants disrupt oestrous cyclicity in the mare.

There is a need for a safe, effective and practical method of oestrus suppression in the mare. The aim of this study was to monitor ovarian activity in mares exposed to either 9.4 or 28.2 mg deslorelin acetate, a GnRH agonist, in the form of a sustained-release implant. Following oestrus synchronisation, mares were randomly assigned to one of three groups (n = 4 per group) and administered either one (Des1 group; 9.4 mg) or three (Des3 group; 28.2 mg) implants of deslorelin acetate (Suprelorin-12, Virbac Australia) or one blank implant (Control group; Virbac Australia). Mares underwent weekly blood sampling for 12 weeks following implant placement (Day 0-Day 84), with transrectal palpation and ultrasonography of the reproductive tract at all sampling timepoints except Days 56, 70 and 77. All mares showed baseline serum progesterone concentrations (SPC; ≤1.3 nmol/L or 0.4 ng/ml) on Day 0. Cycling Control mares showed typical oestrous cyclicity characterised by peaks and troughs in SPC over time. Four of eight treated mares demonstrated a sustained elevation in SPC after the initial ovulation after implant placement; SPC declined to baseline levels (Des1 group; 2 mares) or remained elevated (Des3 group; 2 mares) at the final sampling timepoint on Day 84. Oestrous cyclicity was erratic in three of the remaining four treated mares. In total, 87.5% (7 of 8) of treated mares showed atypical oestrous cyclicity after implant placement. These results suggest that deslorelin acetate disrupts oestrous cyclicity in the mare, which warrants further research.

Comparison of the initial ovarian response, the synchrony of oestrus and ovulation and chronic stress response after administration of 100 or 250 µg of GnRH to randomly cycling Bos indicus cattle.

OBJECTIVE: This study investigated the effects of administering saline, 100 or 250 µg of gonadotrophin releasing hormone (GnRH) on ovarian response, synchrony of oestrus and ovulation and chronic stress response in Bos indicus cattle. DESIGN: Randomised control. METHODS: Animals were either left untreated (n = 20) or on day 0 treated with an intravaginal progesterone releasing device and either saline (n = 24), 100 µg (n = 35), or 250 (n = 35) µg of GnRH, intramuscular (IM). Blood was sampled 1.4 h after administration of treatment to monitor concentrations of luteinising hormone (LH) and P4 in serum and again 5 days later. On day 5 intravaginal P4 releasing device were removed, cloprostenol was administered IM and again 8 h later. Oestrus and ovulation were then monitored with ultrasonography for 6.5 days. Hair was clipped on day 55 for analysis of hair cortisol concentrations (HCC). RESULTS: No significant differences were found between Saline and GnRH treatments in the odds of inducing a new corpus luteum (CL) and the synchrony of oestrus or ovulation. HCC did not differ significantly between treatments. Mean concentrations of LH in serum on day 0 were less in the Saline compared to 100 and 250 µg GnRH treatments but did not differ between different doses of GnRH. CONCLUSION: Mean concentrations of LH and the odds of inducing a new CL were not increased after administering 250 µg compared to 100 µg of GnRH. Animal handling events in the study did not influence HCC. Further research is needed to better optimise responses to GnRH in B. indicus cattle.

The contraceptive efficacy of a self-assembling intra-uterine device in domestic mares.

OBJECTIVE: The primary aim of this study was to investigate the contraceptive efficacy of a self-assembling uterine device (iUPOD™) in the mare. In addition, the effects of iUPODs on oestrous cyclicity, uterine health and circulating concentrations of cortisol were evaluated. METHODS: Domestic mares underwent oestrous monitoring and artificial insemination. After subsequent ovulation, mares underwent either placement (n = 7) or sham placement (n = 7; controls) of an iUPOD device. Devices were left in place for at least 3 months. Pregnancy diagnoses were carried out 14 days post-ovulation, with any pregnancies terminated at 28 days post-ovulation. All mares underwent weekly blood sampling with or without reproductive examinations throughout the study. Towards the end of the study, multiple serum samples collected over three consecutive days were analysed for concentrations of cortisol. Endometrial biopsies were collected before artificial insemination and during the subsequent breeding season. Endometrial cytology and bacterial cultures were performed before device removal (iUPOD mares) or at the end of the study (control mares). RESULTS: Pregnancies were diagnosed in 0 of 7 iUPOD mares versus 7 of 7 control mares. Placement of iUPODs was associated with extended luteal phases and variable accumulations of intra-uterine fluid. Bacterial culture results suggested that the mild endometritis associated with iUPODs was sterile in six of seven mares. Short-term placement of iUPODs had no detrimental effects on endometrial architecture. Mean serum cortisol concentrations were significantly lower in iUPOD mares than control mares. CONCLUSION: iUPODs represent a promising means of fertility control in the mare.

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.

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.

Dynamics of African horse sickness virus nucleic acid and antibody in horses following immunization with a commercial polyvalent live attenuated vaccine.

African horse sickness (AHS) is a fatal disease of equids relevant to the global equine industry. Detection of AHS virus (AHSV) during outbreaks has become more rapid and efficient with the advent of group specific reverse transcriptase quantitative polymerase chain reaction (GS RT-qPCR) assays to detect AHSV nucleic acid. Use of GS RT-qPCR together with recently described type specific (TS RT-qPCR) assays cannot only expedite diagnosis of AHS but also facilitate further evaluation of the dynamics of AHSV infection in the equine host. A potential limitation to the application of these assays is that they detect viral nucleic acid originating from any AHS live attenuated vaccine (LAV), which is the vaccine type routinely administered to horses in South Africa. The aim of this study was to contrast the dynamics and duration of the RNAaemia to the serological responses of horses following immunization with a commercial polyvalent AHSV-LAV using GS and TS RT-qPCR assays and serum neutralisation tests. The results of the study showed extended RNAemia in vaccinated horses, and that more horses tested positive on GS RT-qPCR with lower Cq values after receiving the AHSV-LAV containing types 1, 3 and 4 prior to the vaccine containing types 2, 6, 7 and 8, rather than when the vaccine combinations were reversed. Furthermore, lower Cq values were obtained when vaccines were administered 4weeks apart as compared with a longer interval or 12weeks apart. These findings are of particular relevance in regions where AHSV-LAVs are used as the use of these vaccines may complicate the accurate interpretation of diagnostic testing results.

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.

The first case of genetically confirmed monozygotic twinning in the dog.

Monozygotic twinning has not previously been genetically confirmed in the dog. This case report describes the finding of two viable male monozygotic foetuses within one placental site during caesarean section. Their umbilical cords attached to a single placenta. Genetic profiling using a total of 38 microsatellite markers, as well as amelogenin and SRY for sex determination, revealed identical DNA profiles, whether derived from blood or tissue (buccal swabs) samples. To the best of our knowledge, this is the first report of monozygotic twinning in the dog confirmed using DNA profiling.

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.

African horse sickness in naturally infected, immunised horses.

To determine whether subclinical cases, together with clinical cases, of African horse sickness (AHS) occur in immunised horses in field conditions, whole blood samples were collected and rectal temperatures recorded weekly from 50 Nooitgedacht ponies resident in open camps at the Faculty of Veterinary Science, University of Pretoria, Onderstepoort, during 2008-2010. The samples were tested for the presence of African horse sickness virus (AHSV) RNA by a recently developed real-time RT-PCR. It was shown that 16% of immunised horses in an AHS endemic area were infected with AHSV over a 2 year period, with half of these (8%) being subclinically infected. The potential impact of such cases on the epidemiology of AHS warrants further investigation.