Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions

peer reviewedStallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners’ concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by elec- tro-ejaculation under anesthesia, or after pharmacological induction. The collection method influ- ences the semen volume and concentration, while the total sperm number depends on the testicular production and collection frequency. In the seminal plasma, acidity, pro-oxidant activity, and some enzymes have repercussions for the semen quality and its conservation. Moreover, non-sperm cells of seminal plasma may impact semen conservation. Motility analysis remains a core parameter, as it is associated with fresh or frozen dose fertility. Computer-assisted motility analyzers have im- proved repeatability, but the reproducibility between laboratories depends on the settings that are used. Morphology analysis showing spermatozoa defects is useful to understand production and maturation abnormalities. Staining of the spermatozoa is used to evaluate viability, but recent ad- vances in flow cytometry and in fluorochromes enable an evaluation of multiple intracellular pa- rameters. Spermatozoa protein expression already has clinical applications, for example, as a fertil- ity and freezing ability predictor. At present, stallion semen analysis ranges from macroscopic eval- uation to assessing spermatozoa proteins. However, clinically, all these data may not be relevant, and the lack of standardization may complicate their interpretation

Comparison of combined thickness of the uterus and the placenta between 2 breeds of mares and their potential relationship with estrogens during normal pregnancy.

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Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals

peer reviewedMany fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry

OPTIMIZING DRONE RAISING IN BELGIUM AND SEMEN CRYOPRESERVATION TECHNIQUES

La conservation de la semence de faux bourdons s’inscrit dans un intérêt scientifique mondial croissant. Il est crucial en termes de sélection et de conservation de la biodiversité de pouvoir conserver un potentiel génétique exploitable et mondialement transportable. Afin d’améliorer la qualité de la semence en post-décongélation, certains paramètres restent à évaluer. L’influence de l’âge au moment de la collecte est l’un des critères. Pour l’évaluer il est important de développer une méthode d’élevage permettant d’obtenir un grand nombre de mâles d’âge connu tout en conservant l’équilibre de la colonie. Ce travail décrit le développement d’une méthode d’élevage de mâles encagés d’âge connus. Celle-ci remplit l’objectif d’équilibre stable de la colonie. Le travail rapporte également les premiers essais de congélation de semence. La cytométrie en flux peut évaluer différents critères de qualité de semence dans de nombreuses espèces domestiques, cette technique est utilisée chez le faux bourdon pour évaluer la viabilité en utilisant notamment les marqueurs SYBR-14 et propidium iodide. Les résultats sont difficiles à interpréter et la validation est en cours grâce aux images d’épifluorescence. Il est nécessaire de confirmer les résultats d’élevage en répétant la méthode la saison prochaine et évaluer la qualité de la semence de décongélation.Honeybee semen conservation is of worldwide scientific interest with increasing urgency to improve results in order to conserve biodiversity. Gamete cryopreservation from selected lines is of growing interest also. Indeed, selection of bees tolerant to varroa is one of the main actual objectives in honeybee research. Cryobanking of these gametes will be exploitable internationally by simple transport. In order to improve cryopreservation, different parameters are studied such as influence of drone age. To study this criterion, we need a method to raise large numbers of drones of known age without impairing the colony’s stability. We also need objective characteristics of semen quality. One of these is semen viability studied by epifluorescence or flow cytometry using SYBR-14 and propidium iodide dyes. This report describes the development of this technique and first attempts of cryopreservation. It also details the first steps of validation of the fluorescent supravital staining by flow cytometry as a reference to test new dyes. We can conclude that the rearing method allows to raise large numbers of drones of known age and needs to be tested again during the next breeding season and that the flow cytometry technique needs to be further validated with comparison to epifluorescence microscopy

Melatonin implants to control estrus in Belgian breeding queens

Introduction and aim. Seasonal anestrus in the queen is associated with high levels of melatonin. The use of subcutaneous melatonin implants developed for ovine estrus stimulation (Melovine® 18mg; CEVA santé animale) has been tested in queens with variable results depending on environmental conditions. The aim of this clinical report is to evaluate the use of melatonin implants in field conditions in Belgian catteries. Materials and methods. 13 melatonin implants (Melovine® 18mg) were injected subcutaneously in the neck of 12 pubescent and 1 prepubescent queens. All queens were in interestrus or in prepubescent anestrus. Absence of estrus was confirmed by vaginal smear stained with a modified Harris-Shorr (Kit Diag-Oestro®, RAL Diagnostics, France). Queens were considered not in estrus if less than 70% of the cells were keratinized. The owner was asked to report signs of estrus during the 10 first days following the injection and the duration of the subsequent interestrus. Observed matings after return to cyclicity and potential pregnancies were also recorded. Implants were considered efficacious if interestrus lasted more than 6 weeks. Results. Pubescent queens: implants were efficacious in 11/12 queens with a mean duration of action (from implantation to estrus) of 106 +/- 40 days. 4/11 presented mild signs of estrus with meowing for 2 to 3 days within 10 days post implantation but none was mated during these periods. The queen that failed to respond had a vaginal smear of 70% of keratinized cells at the time of implantation with a history of a 3 week-long estrus ending the day before. It was however decided to try implantation on this unclear case. The next estrus started the very next day to last for another 3 weeks. The breeder then decided to mate her and she got pregnant. Following this pregnancy, this same queen was implanted 3 times during the breeding season on a confirmed absence of estrus and the mean duration of the provoked interestrus was 55 +/- 8 days. One fourth implantation was done 6 weeks post-partum, in October, while she was lactating and a prolonged anestrus until mid-February (130.9 days) was observed. There was no interference with milk production and the kittens were weaned 3 weeks after implantation. A different queen was also implanted in October, when natural secretion of melatonin increases, and anestrus was also prolonged until mid-February. These two cases with increased endogenous secretion of melatonin may have affected the overall efficacy of implants to postpone estrus we report here. However, 3 other queens resumed cyclicity after implantation in November and December. After cyclicity resumption, 6 queens were successfully mated while another one, where no successful cover was observed, failed to get pregnant. That queen was subsequently neutered. The prepubescent queen was 6 months old when implanted, estrus appeared 52 days after. Conclusions. Melovine® was safely used on one lactating queen but its innocuousness should be confirmed by larger studies. Our results also suggest that melatonin should be considered to prolongate post-partum anestrus. Mean duration of action is comparable to that previously reported in French and Italian catteries. Some queens showed signs of estrus in November and December while they should have been in seasonal anestrus. Late in the year implantation of melatonin may provide a promising tool to obtain seasonal anestrus in queens that cycle all year round. In our field conditions, the one prepubescent implanted queen showed first estrus signs at the age of 7.5 months, which is normal for puberty. This is in agreement with results obtained under controlled conditions, where no significant delay in first estrus occurrence was observed. Our results support the observation that fertility does not appear to be compromised by melatonin implants, making it a viable option for cyclicity control in catteries

Optimizing drone raising and marking techniques in Belgium: a report.

Research on drone semen freezing requires very large numbers of drones, which can be very challenging. No established technique to supply these large numbers has been clearly described. We report our attempts to reach sufficient numbers of drones while maintaining a viable balance in the breeding system under Belgian beekeeping conditions. Controlling the age of drones can also be an issue. We report marking drones every 24h for up to 16days using one different color POSCA® Marker per day. Colored drones were well tolerated by the workers. Drones frames were introduced in a strong colony in a Dadant 10 frames hive for the queen to lay eggs. 24h before first hatching the frames were placed in a Dadant 6 frames hive and 2 different caging techniques were tested. Technique 1: Males were kept on a frame in a cage that was opened for daily marking. We observed that the cage quickly got overcrowded and drones tended to escape or get crushed and killed during the manipulations. The technique was then slightly modified and drones were individually collected from the frame, marked and then placed on a caged workers frame. This, however, was associated with too a high number of drones flying away during the manipulation and was abandoned. Technique 2: A maximum of 3 frames of males, 1 of workers and 1 of food were transferred into the body of a Dadant 6 frames hive placed on top of an empty super with a queen excluder between them. Date of birth was assessed by daily marking of the emerged drones. Drones were at first well tolerated but after one month they were chased out of the hive and killed by the workers. Drones raised in the small hive were smaller than drones that escaped and were raised by the nearby colonies. They also had diarrhea and almost no semen could consequently be collected. We conclude that raising large numbers of drones in surrogate hives is suboptimal. An alternative where drones are raised in their home hive and kept in small groups in small cages should be investigated.FreezeBe

Evaluation of short-term storage of canine semen at room temperature and at 37°C – Preliminary study

peer reviewedKeeping dog raw semen at room temperature would prove convenient for practitioners, but validated information regarding short-term storage of semen is scarce. 0.4 ml of sperm rich fraction from 10 healthy beagles (age 1 to 7 years) were collected by digital manipulation, pooled and divided into 3 groups: (1) dilution 1:3 with homemade Tris-fructose 20% egg yolk, (2) dilution 1:3 with a commercial egg yolk-based extender (CaniXCell®) and (3) a sample left undiluted. One half of samples was stored at room-temperature (RT, 22°C) in the dark and the other half in the incubator at 37°C. Samples were submitted to microscopy and CASA (IVOS IITM), at 6 time points post-collection: 0, 12, 18, 24, 36 and 48h. Progressive Motility (PM) was assessed at all times and percentage of wobble movements was evaluated, as a marker of hyperactivation, if PM was higher than 30%. The initial PM at T0 averaged 85%. PM dramatically dropped below 6% in all groups at 37°C and further evaluation was discontinued. At RT, PM was still higher than 70% and 50% at 12 and 24 hours respectively in all groups. Surprisingly, non-diluted semen still showed 18% of MM after 48h, while it was nil in both diluted groups. Percentage of wobble movements was significantly lower at 24h in non-diluted semen than in diluted groups. The premature hyperactivation of diluted semen probably explains the shorter survival time in these samples. No bacterial contamination was observed under microscopy throughout the experiment. These results suggest that short-term storage of good quality undiluted semen at room temperature in the dark may be adequate to inseminate a bitch within 12 hours

Emergency C-section and pre-natal maternal corticosteroid therapy: Three cases in pre-term brachycephalic dogs

Introduction: C-section performed before 62 days post LH surge is associated with a high risk of neonatal mortality due to fetal immaturity. Among other factors, increase of cortisol concentrations pre-partum plays an important role in final development of fetal pulmonary, renal, liver and gastro-intestinal systems (1). Pre-natal corticosteroid therapy has been used since 1972 in human medicine to prevent or, at least to reduce, respiratory complications in pre-term infants. This treatment is associated with a significant reduction of morbidity and mortality resulting from Neonatal Respiratory Distress Syndrome (2). Regazzi et al, Zaremba et al and Rider et al have confirmed the same results in dogs, calves and rabbits. Foetal lung development can be divided in five phases: embryonary, pseudoglandular, canalicular, saccular and alveolar. Pulmonary surfactant is secreted by pneumocytes II during the saccular phase. In 2009, Sipriani et al studied the development of the pulmonary structure throughout pregnancy in dogs. They reported that the saccular phase begins at the earliest 57 days post fertilization; thereby underlying the non-viability of younger puppies. Here we describe three cases of pre-term C-sections with pre-natal corticosteroid therapy. Clinical cases: Two English Bulldogs and one Chihuahua underwent emergency C-sections. The three dogs had been trans-cervically inseminated once with fresh semen. They were followed during the estrus cycle, by vaginal smear and progesterone (P4) assay. Abdominal ultrasound with measurement of the inner chorionic cavity confirmed that the day of fertilization matched with the day of insemination (3). The two Bulldogs and the Chihuahua were presented for anorexia, dyspnea and exhaustion 56 days and 54 days post insemination respectively. Progesterone levels were around 10 ng/ml. The 3 dogs underwent the same surgical and anesthetic protocol. Two hours prior to surgery: perfusion with Hartmann + Glucose 5% solution, injection of prednisolone IM (0.5mg/kg), metoclopramide SC (0.5mg/kg) and amoxicillin clavulanic acid SC (8.75 mg/kg). Induction was achieved with dexmedetomidine (375μg/m2) and alfaxan IV (0,2mg/kg) maintenance with Isoflurane. The linea alba was locally anesthetized with lidocaine. Methadone (0.1 mg/kg) was administered IV at the time of delivery and again upon waking. The first Bulldog gave birth to 8 puppies: 3 had a cleft palate and were euthanized. One died after 4 days from respiratory distress. The four remaining ones survived. The second Bulldog delivered 10 puppies: 1 water puppy and 1 with cleft palate that were euthanized, 2 mummies. Out of the 6 other puppies only one died at 15 days from respiratory distress. The Chihuahua gave birth to 3 puppies who survived without complication despite their high degree of immaturity (hairless) and were sent back home. Discussion: Pre-natal corticosteroid therapy has been shown to improve neonatal viability in several studies. Betamethasone injected 2 days before surgery has been proposed as the treatment of choice. However, Vannuchi et al reported in 2012 a suppression of the fetal and maternal adrenal cortex as well as a premature labor with an administration of betamethasone at 0.5mg/kg. Maternal treatment with prednisolone two hours before surgery should be investigated in order to measure its impact on the fetal and maternal cortisol levels in addition to its effect on surfactant production. Most studies on pre-natal corticosteroid therapy in dogs define the prematurity of the fetuses based on progesterone levels, the predicted LH surge and estimation of the ovulation 2 to 3 days post LH surge. In order to increase the accuracy of the gestational age, we combined progesterone levels, vaginal smear and US measurements. We report here three cases of emergency C-section with viable fetuses at 56 and 54 days post fertilization. We conclude that prednisolone injection 2 hours prior to surgery could represent an interesting protocol to increase neonatal viability and should be further investigated. References: 1) Fowden et al. Proc Nutr Soc. 1998; 57: 113-122. 2) Regazzi et al., Theriogenology 2017; 97:179-185. 3) Lopate. Theriogenology. 2008; 70: 397-402

Computed tomography and ultrasound examination of subcapsular prostatic cyst in a fiveyear-old dog with severe benign prostatic hyperplasia.

Clinical case: A five-year-old Bullmastiff dog was initially presented for staging of an extra skeletal osteosarcoma on the right hip. The dog had a history of intermittent haematuria for months that had never been investigated. Abdominal computed tomographic examination (CT) incidentally revealed an enlarged (8x7.6x5cm) and heterogeneous prostate with contrast enhancement and periprostatic fat stranding. Several hypoattenuating areas were observed in the parenchyma and its periphery, some of them outlined by a rim enhancement on post-contrast scan. Prostatitis and prostatic abscesses associated with secondary steatitis were suspected. Bilateral mild medial iliac lymphadenopathy was present. Ultrasound examination (US) showed an enlarged (7.6cm width, 4cm height in cross section), bilobed prostate. Anechoic dilated ducts radiated from the middle of the gland and connected with an anechoic, septated and irregular-shaped area with distal enhancement between the parenchyma and the distended prostatic capsule of the right lobe. The surrounding fat was hyperechoic, suggesting secondary steatitis. No abnormality was found in the testicles. Cytology from fluid collected by prostatic lavage (PL) showed red blood cells and prostatic cells but no inflammatory cells or bacteria. Urinalysis was within standard limits. Ultrasound-guided fine needle aspiration (US-FNA) of the subcapsular cavity and prostatic parenchyma were performed and 0.2 ml of serous fluid were collected from the cavity. No signs of inflammation were observed on cytological examination. A severe benign prostatic hyperplasia (BPH) and a subcapsular cyst were diagnosed. No US-FNA of the iliac lymph nodes was performed. The dog underwent castration at the same time as surgical removal of the hip mass. Regardless of his BPH, he received cefalexin and carprofen during one week. The dog showed no urinary symptoms during the three weeks after surgery and no ultrasound control was performed. Discussion: Small intraparenchymal cysts are common findings in BPH. Obstruction of ducts by cellular hyperplasia and hypertrophy leads to accumulation of non-inflammatory prostatic secretions within the parenchyma. We report that, while most cysts associated with BPH are located within the parenchyma, they can also collect peripherally between the prostatic parenchyma and the capsule. In this case, CT showed severe enlargement and heterogeneity of the prostate. CT is reported to be more accurate than US for evaluating prostatic size. Prostatic height may have been underestimated by US. Heterogeneous tissue structure and changes in attenuation on CT after contrast agent injection seem to be very sensitive and to detect earlier prostatic changes than US1. However, these features do not specifically differentiate between different prostatic conditions and should not be over-interpreted. Prostatic surrounding reactive fat at US or CT is usually not observed in BPH. Severe enlargement of the prostate could have led to surrounding steatitis. US is the gold standard when investigating the prostate. However, cytology is essential to distinguish between cysts and abscesses2 and to confirm a prostatitis. Cytology of fluid obtained by PL shows a good correlation with histology in case of inflammation. Dilution should be avoided by centrifugation of sample before examination. Contamination by cells of the urinary tract may occur. US-FNA permits to focus on lesion detected by US and shows the strongest correlation with histological diagnosis. Aspiration of fibrotic tissue can lead to poor cellularity.3 In this case, both methods lead to a BHP diagnosis. Increased serum canine prostatic specific esterase (CPSE) concentrations have been reported in dogs affected by HBP, prostatitis or prostatic carcinoma. CPSE could be useful to detect early prostatic disorders but further evaluations are needed to differentiate between prostatic diseases. According to anamnesis and clinical findings, cytology should be performed to refine the imaging diagnosis and propose the most accurate treatment

Surgical removal of a sex-hormone dependant vaginal fibroleiomyosarcoma in a female ferret: a case report.

peer reviewedWhile vaginal smooth muscle tumours have been well described in the dog, they have never been reported in the ferret. A four-year-old intact jill was presented for a mass protruding from the vulva for weeks. The owner reported dysorexia and weight loss unresponsive to NSAIDs and antibiotics. At clinical examination, the ferret was cachectic and presented an alopecia of the tail. A dorsal pediculated white firm mass protruded from a swollen vulva with a purulent discharge. 100UI of hCG were administered to interrupt the oestrus. Three weeks later, she had regained weight, had a better coat, the vulva had reduced in size, but the mass was still present. Under general anaesthesia, the mass was resected through an episiotomy, delicate dissection between the urethral opening and the mass and a ligature around the mass’ pedicle. An ovariohysterectomy was performed. Complete healing of the wounds was observed two weeks later. Unfortunately, no long-term follow-up was available. A low grade fibroleiomyosarcoma, with incomplete surgical excision was diagnosed based on histology and immunohistochemical marking for smooth muscle actin. Leiomyosarcoma have been described in the integument and in the abdominal cavity in the ferret but never in the vagina [1]. Further immunohistolabeling revealed that 80% and 25% of proliferative cells expressed progesterone and oestrogen receptors respectively. Leiomyomas are strongly hormonally dependant and the most frequent vaginal neoplasms in the bitch, and sterilization is considered prophylactic and prevents recurrence if performed at the time of excision of the vaginal mass [2]. Likewise, benefits from sterilization of the jill may be suspected in cases of hormonally dependant vaginal tumours. This is the first report of a sex-hormone dependant vaginal smooth cell tumour in a ferret and its surgical removal. [1] Avallone G, Forlani A, Tecilla M, et al. Neoplastic diseases in the domestic ferret (Mustela putorius furo) in Italy: classification and tissue distribution of 856 cases (2000-2010). BMC Vet Res 2016;12:275. [2] Maxie MG. In: Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. Sixth Edition. Vol. 3, Elsevier Inc 2015: 447-8