Validation of enzyme immunoassays for quantifying sex steroid hormones in tropical screech owls (Megascops choliba).

Androgens and estrogens are steroid hormones that regulate reproductive processes in both males and females. Monitoring plasma levels of these steroids or their metabolites present in feces, offers diagnostic support for assessing the reproductive status of animals. Immunoassays are commonly used methods for quantifying these hormones, but their protocols require species-specific validation to ensure reliability. The objective of this study was to perform analytically and biologically validation of enzyme immunoassay (EIA) kits for measuring testosterone (T), estradiol (E2), faecal androgen metabolites (fAM), and faecal estrogenic metabolites (fEM) in the tropical screech owl (Megascops choliba). Serum and fecal samples were collected from six adult females and six males both before and during breeding season, with males' gonadal activity assessed using electroejaculation (EE). The parallelism test confirmed the immunogenic similarity of the antigens in the estradiol and testosterone standards and the antigens in the serum samples and fecal extracts of M. choliba. Additionally, the EIA kits displayed nearly 100% recovery rates, and showed coefficients of variation ranging from 8% to 14% at the intra-assay level and from 10% to 16% at the inter-assay level, underscoring result reliability and consistency. In males, the highest serum T and fAM levels were recorded concurrently with the presence of spermatozoa in samples collected via EE. Although females did not exhibit oviposition events, significantly higher E2 and fEM levels were observed in August compared to May, suggesting potential seasonal variations in estrogenic hormone production. Fecal androgen and estrogen levels were significantly different between sexes in August, with males having higher fAM and females having higher fEM levels. Overall, the immunoassays validated in this study were found to be efficient in diagnosing reproductive activity in owls.

Case report: Filarial infection of a parti-coloured bat: Litomosa sp. adult worms in abdominal cavity and microfilariae in bat semen.

Background: Filarial infections have been understudied in bats. Likewise, little is known about pathogens associated with the reproductive system in chiropterans. While semen quality is critical for reproductive success, semen-borne pathogens may contribute to reproductive failure. Methods: For the first time we performed electroejaculation and used computer-assisted semen analysis to provide baseline data on semen quality in a parti-coloured bat (Vespertilio murinus). Results: The semen quality values measured in the V. murinus male appeared high (semen concentration = 305.4 × 106/mL; progressive and motile sperm = 46.58 and 60.27%, respectively). As an incidental finding, however, microfilariae were observed in the bat semen examined. At necropsy, eight adult filarial worms, later genetically identified as Litomosa sp., were found in the peritoneal cavity, close to the stomach, of the same particoloured bat male dying as a result of dysmicrobia and haemorrhagic gastroenteritis in a wildlife rescue centre. Histopathology revealed microfilariae in the testicular connective tissue and the epidydimal connective and fat tissues. A PCR assay targeting cytochrome c oxidase subunit 1 confirmed that adult worms from the peritoneal cavity and testicular microfilariae were of the same filarial species. Mildly engorged argasid mite larvae attached to the bat skin proved negative for filarial DNA and the adult filarial worms proved negative for endosymbiont Wolbachia. Conclusion: While the standard filarial life cycle pattern involves a vertebrate definitive host and an invertebrate vector, represented by a blood-sucking ectoparasite, our finding suggests that microfilariae of this nematode species may also be semen-borne, with transmission intensity promoted by the polygynous mating system of vespertilionid bats in which an infected male mates with many females during the autumn swarming. Presence of microfilariae may be expected to decrease semen quality and transmission via this route may challenge the success of reproductive events in females after mating. Further investigation will be necessary to better understand the bat-parasite interaction and the life cycle of this filarial worm.

Effect of Electroejaculation Protocols on Semen Quality and Concentrations of Testosterone, Cortisol, Malondialdehyde, and Creatine Kinase in Captive Bengal Tigers.

The Bengal tiger (Panthera tigris tigris) is critically endangered, so assisted reproductive technologies, including artificial insemination, are important conservation tools. For wild and domestic felids, electroejaculation (EE) is the most common semen collection method, with protocols optimized to obtain sufficient amounts of viable sperm for artificial insemination. However, less attention has been paid to ensuring animal wellbeing during the process. This study examined the effects of three EE protocols (Low, 2-5 volts; Medium, 3-6 volts; High, 4-7 volts) on semen quality, testicular size, serum testosterone, creatine kinase (CK), and malondialdehyde (MDA) concentrations, and serum cortisol as a proxy for stress. Blood samples were collected before, during, and after each EE series. Seminal plasma pH, and sperm motility, viability, and morphology were evaluated after each procedure. Seminal plasma and sperm pellet MDA concentrations were also determined. Primary sperm abnormalities and seminal plasma MDA were higher in the Low compared to Medium and High voltage groups (p < 0.05). Serum CK in the High voltage group increased during the EE series (p < 0.05), suggesting the potential for muscle damage. However, no significant changes were observed for serum cortisol, testosterone, or MDA concentrations. Results suggest the Medium voltage protocol produced good quality samples at lower voltages than the High protocol with no negative effect on muscle function, which might be better for animal welfare.

A novel, patented method for semen collection in dromedary camel (Camelus dromedarius).

In the current article, a developed, patented method denoted the 'Camel Semen Collection Kit-CSCK', was designed to solve the problem of semen collection in dromedary camels. CSCK is composed of three main parts: (1) Semen collection sac: made from supersensitive flexible low-density polyethylene- (LDPE); (2) Metal stainless steel applicator: designed to introduce the collection sac intravaginally and fixate it to the vaginal wall of a female camel through air insufflation; (3) Fixation sticker: a cushion sheet sticker is used to secure the outer portion of the collection sac to the female's perineal area. Semen was collected twice a week from eight dromedary bulls by using electroejaculation (EJ), artificial vagina (AV) and CSCK. Successful semen collections were 81.3%, 84.4% and 43.8% using EJ, CSCK and AV techniques respectively. Semen obtained by EJ technique showed lower semen volume, gross activity, sperm concentration, total sperm motility and percentage of live sperm cells compared to the other two techniques. Semen collected by CSCK showed a longer collection period and higher volume, gross activity, sperm motility and percentage of live spermatozoa and a lower rate of visible contamination compared to AV technique. The advantages and disadvantages of the three techniques were compared and discussed. In conclusion, CSCK represents a practical and easy method to reliably collect high-quality semen from any untrained male dromedary camel and may facilitate the widespread application of assisted reproductive technologies (ARTs) on a large scale in this species.

Administration of carbetocin-a long-acting oxytocin analogue-before sperm collection by transrectal ultrasound-guided massage of the accessory sex glands in bucks (Capra hircus) and ibexes (Capra pyrenaica).

Transrectal ultrasonic-guided massage of the accessory sex glands (TUMASG) is a technique that allows collecting semen requiring few electrical stimuli or even no pulse. A long-acting analogue of oxytocin (carbetocin, 0.1 mg) was i.v. administered before TUMASG in 10 conscious bucks (Experiment 1) and 10 anaesthetized Iberian ibexes (Experiment 2) to shorten the time of semen collection, decrease the number of electrical stimuli and/or improve the semen quality. The ejaculated volume, concentration, quality parameters and kinetics variables of the sperm were determined in fresh semen. The time length of the procedures and the number of electric pulses applied were recorded. Furthermore, stress response indicators (number of vocalizations in Experiment 1; heart and respiratory rates, rectal temperature, cortisol levels, totals proteins and neutrophil-to-lymphocyte ratio in Experiment 2) were documented. In bucks, the administration of carbetocin tended to shorten the time needed for semen collection but no-showed differences in the fresh seminal quality. In the Iberian ibexes, there were no significant differences between groups in the time length of procedures or in the number of animals that ejaculated. Carbetocin administration only reduced the respiratory rate, did it modify fresh semen characteristics in ibexes. In conclusion, the administration of carbetocin did not appear as a useful tool to improve welfare during semen collection with TUMASG or semen quality in conscious bucks and anaesthetized ibexes, having only slight advantages related to the procedure.

Preservation of male fertility in patients undergoing pelvic irradiation.

As the number of cancer survivors increases, so does the demand for preserving male fertility after radiation. It is important for healthcare providers to understand the pathophysiology of radiation-induced testicular injury, the techniques of fertility preservation both before and during radiation, and their role in counseling patients on the risks to their fertility and the means of mitigating these risks. Impaired spermatogenesis is a known testicular toxicity of radiation in both the acute and the late settings, as rapidly dividing spermatogonial germ cells are exquisitely sensitive to irradiation. The threshold for spermatogonial injury and subsequent impairment in spermatogenesis is ~ 0.1 Gy and the severity of gonadal injury is highly dose-dependent. Total doses < 4 Gy may allow for recovery of spermatogenesis and fertility potential, but with larger doses, recovery may be protracted or impossible. All patients undergoing gonadotoxic radiation therapy should be counseled on the possibility of future infertility, offered the opportunity for semen cryopreservation, and offered referral to a fertility specialist. In addition to this, every effort should be made to shield the testes (if not expected to contain tumor) during therapy.

Pharmacological semen collection in domestic and wild canids and felids: literature review.

Semen collection methods vary greatly and rely on the practitioner's expertise, available materials, and the specific behavioral traits of the male animals involved. When it comes to domestic cats, wild felids, and canids, semen collection is particularly challenging. Thus, given the difficulty of semen collection in these species, pharmacological semen collection (PSC) stands out since it is a quick and straightforward method that does not require specific equipment. The PSC consists of administering α2-adrenergic receptor agonist drugs, mainly medetomidine, and dexmedetomidine, aiming semen release into the urethra with posterior urethral catheterization and sperm recovery. This technique was primarily described in domestic cats and wild felids, and despite the decreased seminal volume, the retrieved semen is highly concentrated and presents good quality. However, further studies are required to optimize semen collection in domestic dogs and wild canids. Therefore, the purpose of this review is to provide a comprehensive overview of the research developed on pharmacological semen collection (PSC) in the past few decades. The objective is to equip professionals with the essential knowledge required for the efficient application of this technique in both domestic and wild canids and felids and to make a valuable contribution to conservation efforts and the preservation of biodiversity, aligning with the principles of One Conservation.

Case Studies in Polar Bear (Ursus maritimus) Sperm Collection and Cryopreservation Techniques.

Assisted reproductive technologies can aid conservation efforts via support of ex situ population management and preservation of genetic material. Data from 38 sperm collection attempts from 17 polar bears (1-5 procedures/bear) were evaluated. Sample collections were attempted via electroejaculation (EEJ; n = 6), urethral catheterization (UC; n = 25), or sperm rescue (SR; n = 7) during the breeding season (Jan. 1-May 21; n = 27) and nonbreeding season (May 22-Dec. 31; n = 11). Sperm retrieval was successful in 1 EEJ (16.7%), 18 UC (72.0%) and 4 SR (57.1%) collections. Initial sperm motility and viability were 50.0% and 77.0% for EEJ, 64.3 ± 7.4% and 80.9 ± 3.8% for UC, and 56.7 ± 8.8% and 80.5 ± 0.5% for SR. UC and SR were more likely to be successful during the breeding season (84.2-100%) than the nonbreeding season (25.0-33.3%). Testicular tumors were observed in four males (57%) during SR. In total, 13 samples were cryopreserved (n = 1 EEJ, 9 UC, and 3 SR) with egg-yolk-based equine extender (EQ) or OptiXcell (OP). For both extenders, post-thaw motility and viability were reduced by 20-60% and 30-65%, respectively. Further efforts to optimize procedures are warranted, but this summary provides data useful for enhancing the success of polar bear sperm collection and cryopreservation.

Expression of oxytocin receptors and oxytocin assisted electroejaculation in the domestic cat (Felis catus).

Oxytocin is a peptide hormone that mainly functions to control the contractility of smooth muscles and sex-related steroidogenesis in male reproductive tracts. However, specific information concerning this hormone in controlling the reproductive organs of cats is limited. This study aimed to investigate the expression of oxytocin receptors (OTRs) and their signal mediator via prostacyclin synthase (PTGIS) in reproductive structures following oxytocin assisted electroejaculation. In Experiment 1, the testis, cauda epididymis and vas deferens from five cats were examined by immunohistochemistry and quantitative polymerase chain reaction in order to study the responses of OTR and PTGIS mRNA to oxytocin injection. Experiment 2 examined the effect of oxytocin administration prior to electroejaculation on ejaculate characteristics and sperm quality in terms of motility, viability and fertilizing ability. Immunohistochemistry revealed the expression of OTRs in Leydig's, peritubular myoid cells and some spermatogenic cells. The expression was found in the epithelium and smooth muscle of the epididymis and vas deferens. After oxytocin administration, the OTR mRNA was upregulated in the epididymis (p > .05) and vas deferens (p = .01). The expression level of PTGIS mRNA increased in the response to oxytocin treatment only for the vas deferens (p > .05). Oxytocin treatment before electroejaculation resulted in an approximately twofold increase in sperm concentration and total sperm output/ejaculate, while this intervention did not significantly affect ejaculate volume, sperm quality or fertilizing ability. This study concluded that the oxytocin cascade is locally present in the reproductive structures and plays a role in promoting sperm delivery during electroejaculation in cats.

Semen collection by urethral catheterization and electro-ejaculation with different voltages, and the effect of holding temperature and cooling rate before cryopreservation on semen quality in the Japanese macaque (Macaca fuscata).

In the Japanese macaque, semen has been collected by electro-ejaculation (EE), using the higher voltage stimuli compared to other species including genus Macaca. Semen coagulates immediately after ejaculation, which makes difficult to produce high-quality semen for artificial insemination. Recently, semen collection using urethral catheterization (UC) has been reported in carnivore and this technique may allow semen collection without coagulation in a less invasive manner. Further, the temporal preservation temperature and cooling rate of semen during cryopreservation affect post thawing sperm quality. In this study, to improve semen quality and quantity, as well as the animal welfare, semen collection was performed by EE with high (5-15 V) or low (3-6 V) voltage, UC and a combination of the two (EE-UC). It has been suggested that a high voltage is necessary for semen collection, but 10 V stimulation was effective enough and 15 V is for additional sperm collection. Also, liquid semen was collected by EE-UC and this could increase the total number of sperm. Further, to improve the post thawing sperm motility, semen was kept at four temperatures (4, 15, 25 and 37°C) for 60 min, and processed with two cooling procedures (slow cooling before second dilution and fast cooling after second dilution). Holding semen at 25°C and fast cooling after the second dilution maintained progressive motile sperm rate. The present results will contribute to the improvement of semen collection and animal welfare of Japanese macaques.

Semen collection, evaluation, and cryopreservation in the bonobo (Pan paniscus).

BACKGROUND: Captive breeding of bonobos (Pan paniscus) has proven to be successful, but maintaining genetic diversity remains a challenge. Cryopreservation of semen is an important potential tool to maintain genetic diversity by preserving current genetic material for future use, as well as facilitating the transport and exchange of genetic material. This study aimed to develop a protocol for semen collection and cryopreservation in the bonobo. Semen was collected from four healthy adult bonobos under general anesthesia during management translocation procedures. Semen collection utilizing urethral catheterization was not successful (n = 1), however, all males (n = 4) responded well to rectal probe electro-ejaculation. Immediately after collection, ejaculates were evaluated for color and admixtures, volume, motility, and concentration. Eosin-Nigrosin staining was prepared to evaluate morphology and viability. Ejaculates were split into two equal volumes and cryopreserved in two different extenders, using a one-step and a two-step approach. Ejaculates were gradually cooled to 4 °C in two hours, subsequently stored in liquid nitrogen vapor for twenty minutes (0.25 ml straws), and finally dropped into liquid nitrogen. RESULTS: Pre-freeze evaluation showed thick, white samples with an average ejaculate volume of 450 µl (100-1000 µl), total motility of 59% (40-80%), viability of 69% (38-85%) and 58% (46-72%) normal spermatozoa. Mainly head (22%) and tail (19%) defects were detected on the Eosin-Nigrosin stain. Ejaculates were highly concentrated, nevertheless, due to the coagulum that caused high viscosity and non-homogenous fractions, only estimations of concentration could be made (1000 million/ml). After 24 h of storage, the post-thaw evaluation showed a loss of quality with an average post-thaw total motility of 15% (5-25%) using the one-step freezing medium, and 19% (5-30%) using the two-step medium. Average post-thaw viability was 15% (4-24%) and 21% (15-29%), respectively. CONCLUSIONS: This report on ejaculates from bonobos obtained by rectal probe electro-ejaculation shows that semen parameters of this species are not completely similar to those of its sibling species, the chimpanzee. Further studies are necessary to develop an optimal protocol for the processing and cryopreservation of bonobo spermatozoa.

Proteomic analysis of koala (phascolarctos cinereus) spermatozoa and prostatic bodies.

The aims of this study were to investigate the proteome of koala spermatozoa and that of the prostatic bodies with which they interact during ejaculation. For this purpose, spermatozoa and prostatic bodies were fractionated from the semen of four male koalas and analysed by HPLC MS/MS. This strategy identified 744 sperm and 1297 prostatic body proteins, which were subsequently attributed to 482 and 776 unique gene products, respectively. Gene ontology curation of the sperm proteome revealed an abundance of proteins mapping to the canonical sirtuin and 14-3-3 signalling pathways. By contrast, protein ubiquitination and unfolded protein response pathways dominated the equivalent analysis of proteins uniquely identified in prostatic bodies. Koala sperm proteins featured an enrichment of those mapping to the functional categories of cellular compromise/inflammatory response, whilst those of the prostatic body revealed an over-representation of molecular chaperone and stress-related proteins. Cross-species comparisons demonstrated that the koala sperm proteome displays greater conservation with that of eutherians (human; 93%) as opposed to reptile (crocodile; 39%) and avian (rooster; 27%) spermatozoa. Together, this work contributes to our overall understanding of the core sperm proteome and has identified biomarkers that may contribute to the exceptional longevity of koala spermatozoa during ex vivo storage.

Effect of fasting prior to electroejaculation on behavioral responses and reproductive parameters in young Simmental bulls.

The objective of the present study was to evaluate the effect of 24 h' fasting prior to semen collection by electroejaculation on behavioral responses, volume of rectal fecal content, bladder size, penis protrusion, erection, ejaculation stimuli, and ejaculate parameters in young Simmental bulls. Twenty-two Simmental beef bulls with an age of 13.2 ±â€¯1.2 mo (mean ±â€¯SD) were used in a prospective randomized blinded controlled cross-over design with two pens fasted for 24 h (n = 9; FAS group), and the other three pens were non-fasted (n = 13; CON group). The bulls were maintained under confined conditions without access to pasture. One week later, the pen treatments were inverted, and semen was collected again under the same conditions and by the same team. The behavioral responses, volume of fecal rectal content, bladder size, as well as the number of stimuli required to obtain penis protrusion, erection, and ejaculation to electroejaculation were measured. The following ejaculate parameters were measured: volume, concentration, spermatozoa motility, and morphology. The behavioral response of the bulls to electroejaculation was not different between the CON group and the FAS group (3.2 ±â€¯0.5 and 3.0 ±â€¯0.7, respectively; P = 0.36). Bladder size was significantly reduced in the FAS group compared with the CON group (2.3 ±â€¯0.8 vs. 2.8 ±â€¯0.9, respectively; P = 0.02). The volume of feces in the rectum was not different between the two groups (CON was 2.3 ±â€¯1.7 and FAS was 3.0 ±â€¯1.8; P = 0.23). Compared with the CON group, the FAS group showed a higher proportion of penis protrusion (100% versus 81.8%, P = 0.10), erection (100% versus 81.8%; P = 0.10), and ejaculation (100% versus 90.9%; P = 0.49). The combined efficiency of penis protrusion, erection, and ejaculation (CE-PPEE) in the FAS group was superior to that of the CON group (P = 0.001). The number of stimuli necessary for penis protrusion, erection, and ejaculation for the CON group were 13.5 ±â€¯3.7, 14.9 ±â€¯3.7, and 20.8 ±â€¯5.8, and they were 15.0 ±â€¯4.2, 16.6 ±â€¯4.2, and 20.2 ±â€¯8.1 for the FAS group. The number of stimuli for penis protrusion (P = 0.09), erection (P = 0.08), and ejaculation (P = 0.77) were no different between the two groups. Ejaculate volume was 4.0 ±â€¯2.6 ml and 4.1 ±â€¯2.3 ml for the CON and FAS groups, respectively (P = 0.90). The motility was 1.4 ±â€¯0.7 and 1.4 ±â€¯0.8 for the CON and FAS groups, respectively (P = 0.72). The concentration of spermatozoa was 336.2 ±â€¯273.1 million and 421.1 ±â€¯300.6 million for the CON and FAS groups, respectively (P = 0.31). The percentage of normal spermatozoa was 50.9 ±â€¯18.8 and 45.6 ±â€¯14.3 for the CON and FAS groups, respectively (P = 0.16). It was concluded that fasting for 24 h prior to semen collection by electroejaculation reduced the bladder size and increased the proportion of bulls with penis protrusion, erection, and ejaculation without any difference detected in behavioral responses, volume of rectal fecal content, and ejaculate parameters.

Electroejaculation in men with spinal cord injury: a step-by-step video demonstration.

OBJECTIVE: To demonstrate the proper technique to perform electroejacuation (EEJ) in men with spinal cord injury (SCI) for the purpose of inducing ejaculation. DESIGN: A video demonstration of the proper technique to perform EEJ in men with SCI using the Seager model 14 electroejaculation machine. SETTING: Major university medical center. PATIENT(S): Men with SCI; institutional review board approval was obtained, and all subjects signed an informed consent form. INTERVENTION(S): Spinal cord injury occurs mostly in young men where the majority suffer from ejaculatory dysfunction. The method of choice to induce ejaculation in penile vibratory stimulation (PVS). PVS is successful in 86% of men with SCI whose level of injury is T10 or rostral. If PVS fails or the level is Caudal to T10, the patient is referred for EEJ. This video will demonstrate the proper technique for successful ejaculation using EEJ. Patients with history of autonomic dysreflexia or their level of injury is T6 or rostral are pretreated with 10-20 mg of nifedipine sublingually 10 minutes before stimulation. The patient is then placed in the lateral decubitus position. The bladder is emptied, and a buffer is instilled. An anoscopy is performed, and a rectal probe is placed. A current is delivered until an antegrade ejaculation is retrieved. A retrograde specimen is collected and examined for sperm identification. Patients with complete SCI (no sensory or motor function is preserved in sacral segments S4-S5) can undergo EEJ without anesthesia. Patients with incomplete SCI (significant nerve sparing or normal sensations) will experience pain during stimulation, and general anesthesia is recommended without the use of muscle relaxing agents. MAIN OUTCOME MEASURE(S): Successful ejaculation after performing EEJ in men with SCI. RESULT(S): Electroejacuation is successful in 95% of men with SCI and in nearly 100% if general anesthesia is used. Outcomes of in vitro fertilization or intracytoplasmic sperm injection after EEJ showed 37.5% pregnancy rate per cycle, 50.0% pregnancy rate per couple, 33.3% live birth rate per cycle, and 43.8% live birth rate per couple. No complications due to EEJ were observed in 953 trials, and none occurred in the patients presented in this video demonstration. CONCLUSION(S): Electroejacuation is a safe and reliable method for induction of ejaculation in men with SCI who fail a trial of PVS.

Spermatic profile of captive giant anteaters (Myrmecophaga tridactyla): Knowing more to preserve better.

The giant anteater (Myrmecophaga tridactyla) is being threatened by natural habitat destruction and fragmentation, illegal hunting and road kills. In this context, the generation of basic information on the reproductive parameters of this species is vital, aiming to improve reproductive management via, amongst others, assisted reproductive technologies. This study aimed to describe the morphological and functional features of semen collected from captive giant anteaters. Electroejaculation was performed in 13 animals housed in zoos located in São Paulo state, Brazil. Semen samples were collected from 13 animals in 16 procedures. Samples were evaluated for volume, motility, vigor, pH, concentration, sperm morphology, and functional tests. The following mean values were obtained: volume 1.28 ± 0.27 mL; motility 28.3 ± 6.2%; vigor 2.4 ± 0.25; concentration 129.4 ± 36.1 × 106 sperm/mL; pH 7.4 ± 0.2. Total acrosome, head, midpiece, and tail sperm abnormalities were 3.2 ± 0.8%, 25.4 ± 3.6%, 20.7 ± 3.2%, and 14.7 ± 2.6%, respectively. Intact acrosome was found in 83.7 ± 3.1% and intact membrane in 81.1 ± 4.0% of all samples collected. Mitochondrial activity was 66.4 ± 6.0% (Class I), 18.7 ± 2.9% (Class II), 8.0 ± 2.0% (Class III), 3.9 ± 1.0% (Class IV), and 3.0 ± 0.9% (Class V). Sperm DNA fragmentation rate was 13.2 ± 3.7%. These results indicated that electroejaculation is a feasible method for semen collection in giant anteaters, allowing a more detailed description of the semen in this species.

Cryopreservation of ferret (Mustela putorius furo) sperm collected by rectal massage and electroejaculation: Comparison of a decelerating and an accelerating freezing rate protocol.

The domestic ferret (Mustela putorius furo) provides a good model for developing new reproductive technologies for use with threatened related species. Such technologies could also be used in the reproductive management of this pet species. The present work reports an improved freezing protocol for ferret sperm. Semen was collected by electroejaculation plus rectal massage (in an attempt to reduce the electrical stimulation necessary) from five adult male ferrets, and then subjected to one of two freezing protocols: (a) from 5 to -35°C at 40°C/min, then from -35 to -65°C at 17°C/min, and finally from -65 to -85°C at 3°C/min-a decelerating freezing rate; and (b) from 5 to - 10°C at 5°C/min, and then from -10 to -130°C at 60°C/min-an accelerating freezing rate. After thawing, the viability and acrosomal integrity of the sperm frozen via the two-step accelerating method were better than those frozen via the three-step decelerating method (43.3 ± 3.5% and 71.2 ± 3.4% compared with 29.7 ± 3.7% and 58.8 ± 3.4% respectively; p < .05). No differences were seen between the methods with respect to sperm motility variables; most sperm (>90%) remained static with both freezing methods. In conclusion, although the method with accelerating freezing rate was associated with better post-thaw sperm viability and acrosome integrity values, neither of the two freezing methods tested provided adequate motility results after thawing. Combining rectal massage with electrical stimuli seemed to reduce the number of the latter required for successful sperm collection.

Case Report: Suppression of Harem Stallion Behavior and Fertility Following Anti-Gonadotropin-Releasing Hormone Vaccination of a Captive Wild Przewalski's Horse (Equus ferus przewalskii).

This report describes an option to modulate the testicular function of wild horses and field methods to assess it. Non-surgical castration of a captive wild Przewalski's stallion with anti-gonadotropin-releasing hormone (GnRH) immunization was performed by sub-cutaneous injection of two doses of 450 µg (3 ml) of GnRH conjugated to diphtheria toxin, further repeated every 6 months. Semen quality was assessed after collection by electro-ejaculation under general anesthesia. Endocrine and behavioral consequences were studied during a 2-year follow-up period. The procedure of electro-ejaculation was safe and effective to collect spermatozoa. Motility was low but was improved by a significant dilution of sample (1v/4v-1v/5v) after collection. Immuno-neutering resulted in a decrease of the total spermatozoa number and motility 1 month after primary vaccination. However, infertility could not yet be guaranteed. Six months post-vaccination, serum testosterone concentrations had decreased and the treated stallion had lost his harem stallion role. Moreover, at the same time, the total spermatozoa number was near zero with no motile spermatozoa, and offspring was no longer observed. As a conclusion, electro-ejaculation under general anesthesia is suitable on wild horses to obtain spermatozoa that should be washed or largely diluted before use for artificial insemination (AI) programs. Anti-GnRH immuno-neutering protocol led to a dramatic decrease of spermatozoa number, motility, and testosterone production. This also induced deep changes in the social structure of the band. Such technique could be considered as an alternative to surgical castration in wild horses.

Novel Low-Voltage Electro-Ejaculation Approach for Sperm Collection from Zoo Captive Lanyu Miniature Pigs (Sus barbatus sumatranus).

Semen collection can be achieved via hand penile massage or rectal stimulation using electro-ejaculation methods. Traditional electro-ejaculation procedure applied relatively high voltage of 3-15 volts with a maximum current of 900 mA. However, these manipulations often result in great stress and discomforts in animals. In this study, we showed low-voltage electro-ejaculation procedure using 2-3 volts with a maximum current of 500 mA can efficiently stimulated ejaculations in zoo captive lanyu miniature pigs with a high success rate of 81.3% (13/16). Besides normal semen properties (semen volume, pH, sperm concentration), we demonstrated that low-voltage electro-ejaculation caused less stress in the animals, and sperm cells obtained via low-voltage electro-ejaculation exhibit low abnormality (10.3%), high viability (84.3%), motility (75.7%), progressive motility (63.7%), and acrosome integrity (88%). However, cryopreservation protocol used in the current study requires further optimization, as sperm mitochondrial function was partially compromised during freezing procedures. Taken together, we demonstrated in this study that a low-voltage electro-ejaculation approach can be used to obtain quality sperm cells from zoo captive lanyu miniature pig with less physical stress during electro-ejaculation procedure.

Urethral catheterization as an alternative method for collecting sperm in the black-footed ferret (Mustela nigripes).

The endangered black-footed ferret (BFF; Mustela nigripes) is an important example of the benefits of assisted reproduction in species conservation with both semen evaluation and artificial insemination using fresh and frozen sperm being successfully incorporated into the breeding program. Currently, electroejaculation (EE) is routinely utilized for semen collection in BFFs, a technique that requires custom equipment and experienced operators, and does not consistently yield viable samples in this species. In this case study, we evaluated the feasibility of urethral catheterization (UC) for semen collection, a method predominately tested in domestic and non-domestic felids, on four occasions (three BFF males). After general anesthesia with a combination of ketamine, midazolam and α2-agonist dexmedetomidine (thought to promote semen release into the urethra), a lightly lubricated, flexible feeding tube was passed into the urethral opening and advanced ~7-8 cm into the urethra. A syringe attached to the feeding tube was used to apply mild negative pressure to collect sperm. Semen samples were successfully collected on all four attempts. Sperm characteristics ranged as follows: 10.5-26.0 × 106 sperm/ml concentration, 50-90% motility and 36-61% normal sperm morphology. This is the first report of the use of UC as a potential alternative to EE in the BFF, a more field-friendly technique that is less invasive and more consistent for obtaining samples free of urine contamination.

Adult and yearling pampas deer stags (Ozotoceros bezoarticus) display mild reproductive seasonal patterns with maximum values in autumn.

The pampas deer is an endangered species, from which reproductive biology little is known. We aimed to describe and compare the reproductive seasonal patterns of adult and yearling pampas deer stags throughout the year, including morphological traits, testosterone concentration, sperm morphology and cryoresistance pattern changes. Six adult (AS) and five yearling (YS) stags were captured with anesthetic darts once in winter, spring, summer and autumn to study morphological variables, serum testosterone and semen. Adult males were heavier, their neck girth tended to be greater and their testosterone concentration was higher than in YS. Animals were heavier in summer and autumn. Neck girth and testosterone concentration were greater in autumn. Scrotal circumference, testicular volume and gonado-somatic index varied with seasons, decreasing from winter to spring, increasing in summer and remaining in greater values in autumn. Sperm quality had maximum values from summer to winter. However, the cryoresistance ratio of motility score was greater in spring. In conclusion, in the captivity conditions, pampas deer stags seems to present a light seasonal reproductive pattern, with maximum testis size, testosterone secretion and fresh semen quality in autumn. Nevertheless, sperm cryoresistance ratio seemed to remain stable along the year. Although YS were still growing, they achieved similar semen quality than AS.