Improved sperm freezing in the endangered African wild dog (Lycaon pictus) using a two-step dilution TRIS-egg yolk extender containing Equex STM

Development of assisted breeding techniques can aid conservation and management of the endangered African wild dog (Lycaon pictus). Previous attempts to freeze sperm from this species has proven unsuccessful with sperm motility dropping to nearly 0% within 2 h of thawing. The aim of this study was to improve the freezing success of African wild dog sperm by testing two routinely used canine cryopreservation protocols. Sperm was frozen from n=3 captive African wild dog males housed at Albuquerque BioPark (Albuquerque, NM, USA) and Binder Park Zoo (Battle Creek, MI, USA) during the breeding season (Aug-Sept 2014). Freshly collected semen samples were evaluated for volume, colour, pH, motility, viability, morphology, sperm number, acrosome status and DNA integrity. Each sample was split and frozen using two different protocols. Protocol 1: semen was diluted with a Tris-egg yolk extender containing 8% glycerol and 20% egg yolk, and slowly cooled from 37°C to 4°C over 2.5 h. The sample was then loaded into 0.25 mL straws, suspended 4 cm over liquid nitrogen vapour for 10 min, then plunged in liquid nitrogen. Protocol 2: semen was first diluted with a Tris-egg yolk extender containing only 3% glycerol and 20% egg yolk, followed by a second extender (same composition) now containing 7% glycerol and 1% Equex STM, added after the 2.5 h refrigeration period. The freezing procedure was the same as Protocol 1. Straws from both protocols were thawed in a 37ᵒC water bath, but Protocol 2 straws were further diluted by with a thawing solution which that consisted of the initial extender solution without glycerol and egg yolk. Sperm were incubated at 37 ᵒC and motility evaluated at 5 min and every 2 h for 8 h after thawing. Viability, morphology and acrosome integrity was evaluated over 6 h and DNA integrity was evaluated immediately post-thaw. Sperm motility declined significantly for both protocols immediately after thawing (fresh 78.9 ± 2.6%; Protocol 1 24.4 ± 5.0%; Protocol 2 36.7 ± 4.2%; P ≤ 0.05). Motility was significantly higher for Protocol 2 from 2 h after thawing (Protocol 1 1.0 ± 0.8%; Protocol 2 30.8 ± 1.9%; P ≤ 0.05) and sperm remained motile for up to 8 h. Sperm frozen with Protocol 2 also had significantly higher viability (Protocol 1 37.0 ± 5.7%; Protocol 2 65.3 ± 9.9%; P ≤ 0.05) and acrosome integrity (Protocol 1 22.8 ± 8.2%; Protocol 2 69.3 ± 8.8%; P ≤ 0.05) immediately after thawing. There was no difference in the proportion of normal morphology or DNA fragmentation between both protocols. Our results demonstrate that using a two-step dilution with TRIS-egg yolk extender containing Equex STM yields greatly improved post-thaw quality and longevity in African wild dog sperm; making it suitable for use in artificial insemination

Dog appeasing pheromone prevents the androgen surge and may reduce contact dominance and active submission after stressful interventions in African wild dogs (Lycaon pictus)

The endangered African wild dog (AWD; Lycaon pictus) is a highly social canid living in packs with a separate male and female hierarchy. Immobilisation, handling and translocations are acute stressors for AWDs, however such interventions are often needed for species management. In addition, new pack formation or temporary pack separation can lead to an increase in intra-pack aggression. The goal of this double-blinded placebo-controlled study conducted in captive zoo populations was to evaluate whether dog appeasing pheromone (DAP) reduces behavioural stress and faecal glucocorticoid metabolite levels (fGCM) normally associated with pack separation, immobilisation and reintroduction (SIR), and to assess whether this reduces aggressive behaviours and faecal androgen metabolite levels (fAM). Four packs (n = 11 males) were treated with DAP and 4 packs (n = 12 males) were treated with a placebo solution, applied at the end of anaesthesia. Behavioural interactions as well as fGCM and fAM were determined from 3 days before until 4–6 days after SIR. No effect of DAP on fGCM was observed, however, fAM increased after SIR in placebo but not DAP treated animals. Moreover, on the day of reintroduction, DAP treated packs tended to have lower rates of contact-dominance and active-submission behaviour, but higher rates of non-contact dominance behaviour. As these effects could decrease the risk of agonistic interactions, DAP may be a useful tool to help manage new pack formations and temporary pack separation

A conservation management toolkit: developing assisted breeding and behavioural management tools for the African wild dog (Lycaon pictus)

The African wild dog (AWD; Lycaon pictus) is endangered with the current population estimated at 6,600 animals, scattered over several subpopulations in Southern and Eastern Africa. They show a complex social structure including a separate male and female hierarchy and a cooperative breeding system where subdominants usually do not breed but help in raising the pups. To maintain a viable captive population and genetic diversity, animals are often translocated between institutions to form a new breeding pack. Similarly, a metapopulation management plan has been introduced in South Africa, involving the reintroduction of AWDs in small protected areas and regular translocations of individuals between subpopulations. However, due to their complex social structure, new pack formations can often lead to aggression between animals resulting in injury or even mortality. Sperm freezing, and development of artificial insemination (AI) techniques, can aid species management and conservation of the AWD. The use of semen cryopreservation and AI could overcome problems of intra-pack aggression associated with new pack formations by supplementing genetic diversity without disrupting existing pack structure; and thereby facilitating captive breeding and metapopulation management. In addition, transporting spermatozoa instead of live animals reduces the risk of disease transmission and has ecological and economic benefits. Sperm from free-roaming males could be used to increase genetic diversity in captivity, avoiding the removal of animals from the wild. Lastly, establishing a sperm bank of genetically valuable animals will provide a genetic back-up of the remaining population, providing a buffer against possible threats. Therefore, the aim of this thesis was to develop assisted breeding and behavioural management techniques to enable the application of AI in this species, through the following objectives: (i) determine the effect of social rank on subordinate male fertility (Chapter 2); (ii) develop a sperm freezing protocol (Chapter 3); (iii) determine if Dog Appeasing Pheromone (DAP) can reduce stress and aggression associated with temporary pack separation (Chapter 4); and (iv) validate the use of behaviour and faecal steroid hormone profiles as a non-invasive way to time the fertile period in AWD females for timed AI (Chapter 5). The study included n=15 males from 5 packs housed in zoological institutions in the US (ABQ, Albuquerque BioPark, Albuquerque, NM; TOP, Topeka Zoo, Topeka, KS; BRK, Brookfield Zoo, Chicago, IL; BIN, Binder Park Zoo, Battle Creek, MI; and OKC, Oklahoma City Zoo, Oklahoma City, OK) and n=13 males and n=3 females from 3 packs housed at Harnas Wildlife Foundation, Gobabis, Namibia (BRU, Brutus pack; PLA, Platform pack; SAN, San pack). Males were immobilised during the pre-breeding season (n=12; ABQ, BRK, BIN, TOP) and breeding season (n=24; ABQ, BRK, BIN, OKC, BRU, PLA, SAN) and male fertility parameters including hormones (faecal androgen - fAM and glucocorticoid metabolite - fGCM concentrations), prostate and testes volume, preputial gland size, semen collection success, and multiple measures of sperm quality were recorded (objective 1). Sperm samples of sufficient quality collected in the breeding season were split and frozen using 2 canine freezing protocols: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution (objective 2). In addition, males from US packs were treated topically either with DAP (n=11; 4 packs) or placebo solution (n=12, 4 packs), applied at the end of anaesthesia prior to reintroduction into the pack (objective 3). Behavioural interactions as well as fGCM and fAM were examined from 3 days before (objective 1) until 4-6 days after immobilisation (objective 3). Moreover, behavioural interactions, faecal progesterone (fPM) and estrogen (fEM) metabolite concentrations were examined for n=3 alpha females during their periovulatory period. Furthermore, each female was immobilised on 2 or 3 occasions at this time to evaluate vulvar size, and serum progesterone and oestrogen levels as well as perform vaginal cytology, vaginoscopy, and ovarian ultrasound (objective 4). Semen could be collected successfully from all alpha males but from only half the subordinate males in the pre-breeding season, with higher urine contamination in lower ranking animals. Fertility parameters did not differ between social ranks, except for a lower sperm progressive motility and normal morphology in subordinates. In the breeding season, preputial gland size increased with social rank, but no difference between ranks was observed in any other parameter, including sperm quality (objective 1). Eight ejaculates of sufficient quality were frozen in the breeding season. Sperm motility persisted for up to 8h after thawing for Protocol 2, while it dropped to nearly 0% after 2h incubation for Protocol 1. In addition, viability and acrosome integrity of spermatozoa were higher for Protocol 2 throughout post-thaw incubation (objective 2). The application of DAP to males at the end of anaesthesia and prior to reintroduction, did not alter the rise in fGCM levels after intervention. However, fAM increased in placebo but was prevented in DAP treated animals. On the day of reintroduction, DAP treated packs tended to show lower rates of contact-dominance and active-submission behaviour, but higher rates of non-contact dominance behaviour (objective 3). In females, late oestrus (fertile period) could be clearly distinguished from early oestrus by an increase in frequency of certain types of behavioural interactions between the alpha male and female (affiliative behaviour, sexual follow, male initiating behaviour, ride-up, and copulation). One female exhibited an anovulatory cycle while the remaining 2 showed a significant 2.5- to 3-fold increase in fPM levels and similar declining fEM levels (after a rise in pro-oestrus) compared to baseline. However, vaginal cytology and vaginoscopy results deviated from patterns seen in domestic dogs, and blood progesterone showed lower levels. Moreover, ovarian structures were difficult to visualise by ultrasound. As such, without frequent sampling, these invasive diagnostic techniques are unsuitable to determine the fertile phase in wild canids such as the African wild dog (objective 4). In conclusion, reproductive suppression of subordinate males appears to be behaviourally mediated, given that AWD males of all social ranks produce semen of similar quality during the breeding season, making them suitable candidates for sperm banking (objective 1). In addition, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it appropriate for sperm banking and artificial insemination initiatives (objective 2). The observed effects of DAP on AWD hormones and behaviour could decrease the risk of agonistic interactions, making it a useful tool to help manage temporary pack separation, needed when performing semen freezing and AI (objective 3). Lastly, distinctive behaviours during late oestrus, together with an increase in faecal progesterone and decline in faecal oestrogen in AWD females, could potentially be used to determine the timing of the fertile period (objective 4). These results combined facilitate sperm banking and the application of AI in the African wild dog, thereby assisting management and conservation of the species

Raw semen concentration directly influences CASA velocity pathways

We observed an influence of the stallion on volume, spermatozoa concentration and all CASA parameters (p<0.0001), which are highly dependant on each other due to geometrical association of these data. That stallion effect may have interfered with the associations we observed as stallions seem to have specific concentration and motility pathways. More studies, with more replicates, will allow comparing results from a same stallion and further establish the correlations we report here

Palatoschisis in the dog: developmental mechanisms and etiology

Palatoschisis is a frequently occurring birth defect in man and domestic animals. It is caused by a failure of the elevation, apposition or fusion of the lateral palatine processes, resulting in the persistence of a slit-like opening between the oral and nasal cavities. Due to swallowing difficulties, this condition eventually leads to severe malnutrition and life-threatening aspiration pneumonia unless adequate treatment is provided. The formation of the palate is the result of a sequence of well-regulated steps. Palatoschisis can result from any interference with local cell proliferation, differentiation and apoptosis, the aberrant production of mucopolysaccharides or interference with the active extension of the neck. It results from a single or combined action of genetic, mechanical and/or environmental teratogenic factors. The complex etiology of a cleft palate, its potential hereditary characteristics and possible association with other congenital defects should be carefully considered prior to any corrective therapy

Squamous metaplasia of the prostate and diffuse alopecia in a 13-year-old castrated dog due to chronic ingestion of exogenous estradiol

Introduction. A 13-year old whippet with a generalized progressive alopecia since 2 years was referred to our clinic to perform a low-dose dexamethasone stimulation test and abdominal ultrasound to exclude or confirm Cushing disease. The dog had previously been treated with trilostane for 4 months without having any positive effects. Earlier blood analysis showed no significantly abnormalities and ACTH stimulation test was negative. Having bilateral cryptorchidism, the dog was castrated at young age. Clinical findings and treatment. Abdominal ultrasound revealed a slightly enlarged right adrenal gland (diameter of 1 cm at the cranial part). The prostate was enlarged and heterogeneous with multiple anechogenic cavities, indicating hormonal stimulation. Craniad the prostate and ventrally of the bladder, a hypoechogenic, oval structure (3 cm by 1.7 cm) could be seen, and was suspected to be a remaining cryptorchide testicle. Fine needle aspiration however showed that the structure contained a thick purulent liquid, with large amounts of polymorhonuclear white blood cells. Blood analysis revealed a leukocytosis (29370/mm3) with neutrophilia (23202/mm3). Low-dose dexamethasone test definitely excluded Cushing disease (basal cortisol levels 2,17 µg/dl; cortisol at 4 and 8 hours < 1 µg/dl). A prostatic wash was performed and revealed a severe prostatitis and presence of keratinized prostatic cells, indicating a squamous metaplasia of the prostate. Measurement of seric LH levels (LH Witness®, Synbiotics) showed an LH concentration lower than 1 ng/ml, indicating a hormonal negative feedback on the hypothalamo-hypophyso-gonadal axis. After a more thorough anamnesis, it became clear that the dog was licking and ingesting a transdermal estradiol containing cream (Estrogel®) from his owner, since two years, causing his symptoms. The dog was put on antibiotic treatment for the prostatitis (enrofloxacine 10 mg/kg SID) and underwent surgery to excise the abscess cranial of the prostate. The two ductuli deferentes were attached to this structure. Histopathological analysis revealed this tissue to be embryological remanents or a morphological anomaly with a urogenital origin. Control after 4 weeks showed that the prostate slightly decreased in volume, however, anechogenic cavities where still present of which one increased in volume. The alopecia was still present as well, both probably due to a prolonged action of the estrogens. Conclusion. Even though injectable estrogen preparations are not longer available in veterinary medicine in Europe, a thorough anamnesis towards other exogenous estrogen sources is still necessary and can reduce the number of supplementary exams performed

A flow cytometric study on the effect of myeloperoxidase on stallion spermatozoal motility and structure

Myeloperoxidase (MPO) is a pro-oxidant enzyme that is normally contained in neutrophils. MPO has recently been associated with keratinized cells and with decreased post-thaw motility in stallion semen [1]. The aim of the study was to determine effects of experimental addition of active MPO on motility, mitochondrial potential, apoptosis induction, membrane and acrosome integrity in equine semen. Three stallions were used and semen was collected four times. Extended (INRA96TM) semen was processed for density gradient centrifugation (Equipure Bottom Layer®) [2]. Purified pellet was re-extended to 100x106spermatozoa/ml in INRA96TM and divided in 3 samples. One sample was used for control and active human MPO (Calbiochem, Merck) was added in the other two samples to final concentration of 5 or 50 ng/ml. After incubation (2 hours, 20°C), motility was analysed with Computer Assisted Semen Analysis (IVOS, Hamilton-Throne, Beverly, MA, USA) and cytometric analyzes were perfomed with EasyCyte (IMV). Mitochondrial potential and apoptosis were assayed using Guava Mitopotential JC-1 and 7-AAD kit (Millipore). Membrane and acrosome integrity were respectively assayed with PI (Propidium Iodide) (Invitrogen) and PNA (Peanut Agglutinin-Fluorescein Iso Thio Cyanate) (Sigma-Aldrich). Statistical differences (p<0.05) were determined using Kruskall-Wallis test. No effect of the stallions was observed on parameters assayed in this study. Unlike total motility, progressive motility was decreased in both MPO concentrations (p<0.001). MPO addition had no effect on membrane and acrosome integrity. No differences were detected for the percentages of spermatozoa having polarised or depolarised mitochondria. Apoptosis, assayed by 7-AAD fluorescence, was not increased by the treatments. Our results agree with previously published effects of in vitro ROS production systems with xanthine oxidase [3], showing an effect on motility but no influence on mitochondria and membrane or acrosome integrity. However, membrane lipoperoxidation was increased by ROS in this study [3], and it could be linked to the impaired motility also observed in our protocol. Further studies with increasing concentrations of added MPO should be conducted to correlate motility with lipoperoxidation. References [1] Ponthier J, Desvals M, Franck T, de la Rebiere de Pouyade G, Spalart M, Palmer E, Serteyn D, Deleuze S. Myeloperoxidase in equine semen: Concentration and Localization during freezing processing. Journal of Equine Veterinary Science 2012;32: 32-37. [2] Edmond AJ, Teague SR, Brinsko SP, Comerford KL, Waite JA, Mancill SS, Love CC, Varner DD. Effect of density-gradient centrifugation on quality and recovery rate of equine spermatozoa. Animal Reproduction Science 2008;107: 318-318. [3] Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MC. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J Androl 2000;21: 895-902.SPERMP