The impact of percutaneous epididymal sperm aspiration on sperm quality in mice.

Abstract: In laboratory mice, sperm quality is usually assessed in spermatozoa collected from the cauda epididymidis of freshly sacrificed males. Percutaneous epididymal sperm aspiration (PESA) is a non-terminal alternative that would allow repeated sperm collection for sperm quality assessment in living males. To test whether PESA is a suitable method to assess sperm quality, we compared sperm traits between samples collected by PESA vs the commonly applied terminal cauda epididymidis dissection. The collected sperm samples were analyzed using computer-assisted sperm analysis and various parameters, including sperm motility, swimming velocity and morphology, were determined. We were able to retrieve motile sperm from all mice using PESA and the terminal cauda epididymidis dissection. Based on computer-assisted sperm analysis, however, sperm motility and swimming velocity were significantly lower after PESA compared to samples obtained by cauda epididymidis dissection. In addition, we found significantly more morphological abnormalities in PESA samples, probably induced as a side effect of the sampling technique. Although sperm samples collected by PESA are successfully used for in vitro fertilization, we cannot recommend PESA as a suitable method to assess sperm quality in mice, since the procedure seems to impair various sperm traits. Lay summary: In mice, sperm quality is usually assessed in sperm collected from the epididymis (organ where ripe sperm is stored) of euthanized males. However, there is one non-terminal and minimal invasive alternative to collect sperm, called percutaneous epididymal sperm aspiration (PESA), which allows repeated sample collections from the same individual. Given that individual sperm quality is variable and can change according to various factors, PESA could allow to track sperm quality over time and would be highly appreciated in different research fields. Here, we tested the suitability of PESA to determine sperm quality by comparing sperm samples collected by PESA vs the commonly applied terminal epididymis dissection. We used computer-assisted sperm analysis to determine various sperm quality traits. Surprisingly, we found that sperm collected by PESA showed significantly reduced motility, swimming velocity and more morphological abnormalities compared to sperm samples collected by epididymis dissection. Thus, we cannot recommend PESA as a suitable method to determine sperm quality traits as the procedure itself seems to affect collected sperm cells.

Eficiência de duas técnicas de recuperação de espermatozoides epididimários de cães e avaliação seminal pós-criopreservação / [Efficiency of two techniques for recovering epididymal spermatozoids from dogs and seminal evaluation post cryopreservation]

O objetivo deste trabalho foi avaliar a recuperação de espermatozoides epididimários de cães castrados, utilizando as técnicas de fluxo retrógrado (FR) e flutuação (FL) em diluidor Tris-gema, antes e após a criopreservação. Foram coletados 30 complexos testículo-epididímos (CTE), sendo 15 para FR e 15 para FL, e, logo após a recuperação dos espermatozoides, foram analisadas as alterações morfológicas nessas células espermáticas. Após a adição do diluidor, foram avaliados os parâmetros de motilidade total (MOT) e vigor (V) espermáticos. O sêmen pós-criopreservado foi submetido ao teste de termorresistência nos tempos T0, T30, T60 e T90 minutos, além da avaliação das membranas plasmática e acrossomal por sondas fluorescentes. Não houve diferença estatística entre as técnicas quanto à MOT e ao vigor no sêmen diluído (FR-MOT: 82,3% e V: 3,4; FL-MOT: 79,6% e V: 3,2) e pós-criopreservado (FR-MOT: 34% e V: 2,8; FL-MOT: 30% e V: 2,7). A partir do T30, houve diferença significativa quanto à MOT e ao vigor nas técnicas utilizadas, e o tempo também prejudicou o acrossoma espermático a partir do T30. Conclui-se que as técnicas de recuperação de espermatozoides epididimários de cães castrados, testadas neste trabalho, podem ser utilizadas para refrigeração e criopreservação de sêmen.(AU)

The objective of this work was to evaluate the recovery of epididymal spermatozoa from castrated dogs using retrograde flow (FL) and flotation (FL) techniques in Tris-egg yolk diluent, before and after cryopreservation. Thirty testicle-epididymal complexes (CTE) were collected, 15 for FR and 15 for FL and soon after spermatozoid recovery, morphological changes in these spermatic cells were analyzed. After addition of the diluent, the parameters of total motility (MOT) and vigor (V) were evaluated. The post-cryopreserved semen was submitted to thermoresistance (TTR) test at T0, T30, T60 and T90 minutes, as well as the plasma and acrosomal membrane evaluation by fluorescent probes. There was no statistically significant difference between techniques tested for MOT and vigor in the diluted semen (FR-MOT: 82.3% and V: 3.4, FL-MOT: 79.6% and V: 3.2) and post-cryopreserved (FR-MOT: 34% and V: 2.8, FL-MOT: 30% and V: 2.7). From the T30 there was a significant difference regarding MOT and vigor in the used techniques, and the time also damaged the spermatic acrosome from the T30. It is concluded that the epididymal spermatozoa recovering techniques from castrated dogs, tested in this study, can be used for semen refrigeration and cryopreservation.(AU)

Semen collection by trans-rectal digital stimulation and insemination campaign in goat.

The overall purpose of this study was to describe a method of semen collection via trans-rectal digital massage (TDM) and to carry out a related fertility trial in Angora goat. Sixteen Angora bucks (ranging 1-4 years) and 28 nulliparous does (1-2 years) were used in this study. Semen samples were collected via trans-rectal massage from 85.71% of the bucks in multiple attempts (18/21). The mean values of volume, pH, mass motility, total motility, concentration, viability, abnormal spermatozoa rate and ejaculation time were 0.64 ± 0.09 ml, 6.3 ± 0.21, 2.7 ± 0.34, 58.18 ± 5.1%, 3.68 ± 0.31 × 109 /ml, 71.38 ± 7.12%, 18.22 ± 2.48% and 3.4 ± 0.33 min respectively. Oestrus was detected with teaser buck and confirmed by using infrared thermography and ultrasonography (US). The success rate of synchronisation was found as 71.4% (20/28). On Day 21, pregnancy diagnosis was performed trans-rectally with US and the pregnancy rate was determined as 78.57% (11/14). TDM method of semen collection seems to be easily applicable to the buck and it could be a good alternative to collect semen as well as its use in artificial insemination campaign. Thermal monitoring is found to be a valuable tool to monitor the response to hormonal driven ovulatory synchronisation in Angora does during timed artificial insemination.

Effect of temperature and time after collection on buck sperm quality.

BACKGROUND: Different parameters are assessed as part of the semen analysis but a standard protocol for evaluation of goat semen is still missing. The aim of this study was to analyse two different factors affecting buck sperm quality in the post-collection period prior to adding the extender. Here we examined the effects of two handling temperatures (20 °C, 37 °C) and various examination time points (3-30 min) after semen collection. RESULTS: Examination time point had a significant influence on raw sperm viability (p < 0.05), motility (p < 0.05) and on semen pH (p < 0.05). The two different handling temperatures had no significant effect on sperm viability (p > 0.05), motility (p > 0.05), with the exception of fast moving sperm (p = 0.04), or on semen pH (p > 0.05). CONCLUSION: Examination time point was identified as factor strongly influencing raw peacock buck semen after collection. Raw goat semen can tolerate room temperatures for at least 10 min without impacting overall semen quality. In order to obtain comparable results, semen samples should always be examined within 10 min after collection.

Protocols using detomidine and oxytocin induce ex copula ejaculation in stallions.

Tricyclic antidepressives, such as imipramine, indirectly induce ejaculation by increasing the noradrenaline concentration, which triggers an α-adrenergic response, whereas α-adrenergic agonists, such as xylazine and detomidine, directly trigger ejaculation by activating the α-1 adrenergic receptors. Furthermore, serum oxytocin concentrations in stallions increase drastically before ejaculation, but decline immediately thereafter, implicating the role of this hormone in emission. The objectives of the present study were to: 1) compare the efficiency of various protocols for inducing ex copula ejaculation in stallions, 2) evaluate the benefits of including oxytocin in the protocols, and 3) compare the semen characteristics of ex copula versus in copula ejaculates. Nine protocols were used to induce ex copula ejaculation using various combinations of xylazine (X; 0.66 mg/kg, iv); oxytocin (O; 20 IU, iv), imipramine (I; 3 mg/kg, orally), and detomidine (D; 0.02 mg/kg, iv). Imipramine was given 2 h prior to the administration of α-adrenergic agonist (detomidine or xylazine) and oxytocin. If ejaculation did not occur within 10 min after treatment with an α-adrenergic agonist, a half-dose of the same product was injected. Twelve sexually mature stallions (6-26 y) were used; 9 of 12 stallions responded to the treatment. Two stallions responded to X or XO, four stallions responded to IX and IXO, one stallion responded to DO, and five responded to IDO. Stallions that responded to detomidine did not respond to xylazine. No stallion ejaculated in response to D, ID, or IO. Erections and masturbation occurred only in imipramine-treated stallions. Sperm quality was similar among all the protocols and was not significantly different from those in in copula ejaculates collected with an artificial vagina. In a separate trial, none of these protocols induced ex copula ejaculation in 2-3 y old stallions. The side effects included sialorrhea after imipramine administration in all the stallions and sedation after administration of xylazine or detomidine. In conclusion, the new protocol, IDO, and the traditional protocol, IX, had similar results, with IDO being a useful alternative protocol in stallions for which IX was not effective. Therefore, attempts using both the protocols are encouraged, as stallions that ejaculated upon administration of detomidine did not ejaculate when xylazine was administered, whereas those that responded to xylazine did not respond to detomidine.

Sperm collection and storage for the sustainable management of amphibian biodiversity.

Current rates of biodiversity loss pose an unprecedented challenge to the conservation community, particularly with amphibians and freshwater fish as the most threatened vertebrates. An increasing number of environmental challenges, including habitat loss, pathogens, and global warming, demand a global response toward the sustainable management of ecosystems and their biodiversity. Conservation Breeding Programs (CBPs) are needed for the sustainable management of amphibian species threatened with extinction. CBPs support species survival while increasing public awareness and political influence. Current CBPs only cater for 10% of the almost 500 amphibian species in need. However, the use of sperm storage to increase efficiency and reliability, along with an increased number of CBPs, offer the potential to significantly reduce species loss. The establishment and refinement of techniques over the last two decades, for the collection and storage of amphibian spermatozoa, gives confidence for their use in CBPs and other biotechnical applications. Cryopreserved spermatozoa has produced breeding pairs of frogs and salamanders and the stage is set for Lifecycle Proof of Concept Programs that use cryopreserved sperm in CBPs along with repopulation, supplementation, and translocation programs. The application of cryopreserved sperm in CBPs, is complimentary to but separate from archival gene banking and general cell and tissue storage. However, where appropriate amphibian sperm banking should be integrated into other global biobanking projects, especially those for fish, and those that include the use of cryopreserved material for genomics and other research. Research over a broader range of amphibian species, and more uniformity in experimental methodology, is needed to inform both theory and application. Genomics is revolutionising our understanding of biological processes and increasingly guiding species conservation through the identification of evolutionary significant units as the conservation focus, and through revealing the intimate relationship between evolutionary history and sperm physiology that ultimately affects the amenability of sperm to refrigerated or frozen storage. In the present review we provide a nascent phylogenetic framework for integration with other research lines to further the potential of amphibian sperm banking.

Sperm handling in aquatic animals for artificial reproduction.

Artificial reproduction involves collection and handling of gametes in a way that secures their quality and maximizes the fertilization outcome. In addition to initial sperm quality, numerous steps can affect the final result of fertilization, from the sperm collection process until gamete mixing (or co-incubation) when the spermatozoon enters or fuses with the oocyte. In this review, we summarize the whole process of sperm handling, from collection until fertilization for fish, penaeid shrimp, bivalve mollusks and marine mammals. To obtain sperm from captive animals, techniques vary widely across taxa, and include stripping by abdominal massage or testis surgical removal in fish, spermatophore collection in penaeid shrimps, gonadal scarification or temperature shock in bivalve mollusks, and voluntary collection via positive reinforcement in mammals. In most cases, special care is needed to avoid contamination by mucus, seawater, urine, or feces that can either activate sperm motility and/or decrease its quality. We also review techniques and extender solutions used for refrigerated storage of sperm across the aforementioned taxa. Finally, we give an overview of the different protocols for in vivo and in vitro fertilization including activation of sperm motility and methods for gamete co-incubation. The present study provides valuable information regarding breeder management either for animal production or species conservation.

Influence of the type of semen and morphology of individual sperm cells on the results of ICSI in domestic cats.

The aim of this study was to analyze the influence of the type of spermatozoa and of different sperm abnormalities on fertilization and embryo development after ICSI in cats. In Exp I, ICSI was performed using urethral or epididymal spermatozoa collected from 7 tomcats. In Exp. II, epididymal spermatozoa from 16 cats were used for ICSI and an epididymal spermatozoon exhibiting no abnormalities or one with an abnormality was microinjected into an oocyte. Exp. I was performed in 14 replicates and Exp. II was performed in 20 replicates. In both experiments the number of cleaved oocytes, the number of embryos at the morula stage and the number of embryos at the blastocyst stage were evaluated at 24 h, and at 6 and 7 days after ICSI, respectively, and compared between experimental groups. No statistically significant differences (P > 0.05) were observed, either for Exp. I or for Exp. II. The average cleavage rate was 60.2%, morula rate 62.3% and blastocyst rate 19.2% in Exp. I and 51.6%, 66.8% and 25.8% in Exp. II, respectively. The study confirmed that both urethral and epididymal spermatozoa can be used for in vitro fertilization in cats and proved the usefulness of the ICSI method in the case of teratozoospermic males. The study showed that even in severe cases, when almost no normal spermatozoa can be found in the semen, it is possible to obtain embryos using abnormal sperm cells with the same chance of success as for normal spermatozoa.

Effectiveness of urethral catheterization under ultrasound guidance for semen collection from Asiatic black bears (Ursus thibetanus).

The Asiatic black bear (ABB; Ursus thibetanus ussuricus) is a globally endangered species, and measures to help increase their population are necessary. For the successful restoration of this species, artificial breeding as well as conservation translocation are considered important. The aims of the present study were to evaluate the feasibility and effectiveness of urethral catheterization (UC), which is effectively used in feline species, for semen collection from ABBs and establish the optimal protocol for semen collection via this technique. Seven clinically healthy, adult male ABBs (age, 6-13 years; weight, 130-180 kg) housed at the Species Restoration Technology Institute, Korea were included in this study. All study procedures were performed during the breeding season (June to August) over 3 consecutive years. Semen samples were collected once or three times from all bears by ultrasound-guided UC or electroejaculation (EE) under general anesthesia, and their characteristics, including sperm motility, were evaluated. The day of semen collection was defined as Day 0. The semen collected by the UC method was stored at 4 °C, and sperm motility was evaluated at the same time every day for 16 days. The successful collection rates for the UC and EE methods were 92.3% and 53.8%, respectively. The sperm concentration (4718.9 ±â€¯1526.1 vs. 185.0 ±â€¯34.2 × 106/ml), total sperm count (1196.6 ±â€¯955.5 vs. 100.9 ±â€¯70.0 × 106), sperm motility score (4.39 ±â€¯0.78 vs. 3.00 ±â€¯1.73), viability (98.2 ±â€¯2.3 vs. 82.7 ±â€¯19.6), and the proportion of spermatozoa with intact acrosomes (92.2% ±â€¯9.3% vs. 75.6% ±â€¯10.6%) were higher with the UC method than with the EE method, whereas the proportion of spermatozoa with an abnormal morphology (23.1% ±â€¯4.6% vs. 45.6% ±â€¯19.5%) was lower with the former than with the latter. Over the course of cool storage, there was an overall decrease in the total motility, progressive motility, and viability, although viability was >50% until Day 10. These findings suggest that ultrasound-guided UC is a useful and feasible tool for the collection of high-quality semen from ABBs. The collected semen remains viable for up to 10 days, with high sperm motility maintained for up to 7 days, when stored at 4 °C.

Effect of length of time post-mortem on quality and freezing capacity of Cantabric chamois (Rupicapra pyrenaica parva) epididymal spermatozoa.

Genome Resource Banks are keystones in the ex-situ conservation of wild species. Post-mortem (PM) collection of epididymal spermatozoa is an opportunistic and valuable source of germplasm, the time from the death of the animal limits its use. Seeking to improve germplasm preservation strategies for the chamois (Rupicapra sp.), the effect of PM time on epididymal sperm quality and freezability was studied using the Cantabrian chamois. Samples were classified according to PM collection time, up to 216 h (refrigerated), and cryopreserved (Tris-citric acid-fructose, 430 mOsm/kg, 15% egg yolk, 8% glycerol; freezing at -20 °C/min). Sperm quality was assessed after recovery and post-thawing (motility by CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). The sperm mass pH and osmolality showed a positive correlation with time. Total sperm motility dropped after 2 days PM, with progressivity and sperm velocities remained similar up to 3 days PM. Sperm freezability was acceptable, with the post-thawing HOST, motility, progressivity, VAP, VCL, VSL and BCF negatively correlating with PM time. Overall, chamois epidydimal samples were not adequate for preservation after 6 days PM. Freezability capacity could make these spermatozoa suitable for specific ART even if kept refrigerated for several days PM.

Optimization of the incubation time and temperature for spermatozoa extraction in freshwater crayfish Pontastacus leptodactylus (Eschscholtz, 1823).

Determination and control of spermatozoa quality in crustacean aquaculture is an important issue for successful and controlled reproduction. Investigation of spermatozoa number in spermatophores is a basic and common parameter for determining the reproductive quality in farmed decapods. In the present study, spermatozoa extraction from spermatophores located in the ductus deferens was conducted in Pontastacus leptodactylus using different incubation times and temperatures. The results indicate that the duration of incubation and temperature affected (P < 0.05) spermatozoa extraction. Greater temperatures (40 and 75 °C) resulted in a reduction (P < 0.05) in number of extracted spermatozoa. In contrast, more spermatozoa were extracted when the 4 and 23 °C temperatures were imposed. After 4 h of incubation, the number of extracted spermatozoa were greatest in number at 23 °C. In conclusion, the greater numbers of crayfish spermatozoa can be obtained when the ductus deferens containing spermatophores is incubated at 23 °C for 4 h as compared with other temperatures and incubation durations. The results of present study are useful for assessing spermatozoa quality in aquaculture as well as the extraction of spermatozoa for research purposes.

Modeling of behavioral responses for successful selection of easy-to-train rams for semen collection with an artificial vagina.

The aim of this study was to analyze the reproductive behavioral responses in Australian Merino rams, to identify those related to a faster training for semen collection with an artificial vagina. Eight Australian Merino rams, aged 1.5 years and with no prior sexual experience, were randomly selected from an extensively grazed flock. One immobilized ewe with no hormone stimulation was used for rams to sexually interact and mount. The frequencies of approaching, sniffing, flehmen, pushing, pawing with chin resting, and tongue flicking were recorded during eight training and three post-training assessments periods. In addition, the duration of sniffing and flehmen responses, as well as the time from when the ram started to approach the ewe until the mount with ejaculation (completed mount) were recorded. Descriptive, correlation, and modeling analyses were performed. Amongst the rams, four mounted the ewe and ejaculated for the first time during the training phase, and three mounted and ejaculated for the first time after the training phase. The remaining ram mounted the ewe and ejaculated for the first time during the post-training evaluation in the following year. A great variability in the behavior repertoire was observed among rams. The correlation analysis indicated that the completed mount was associated with the behaviors during the approaching response. The expression of the sniffing response decreased between the training phase and post-training evaluation, while the responses of pushing the ewe and tongue flicking ceased to occur. Pawing the side of the ewe with the chin resting on the back of the ewe and flehmen responses, however, continued between the training and post-training phases. This led to a decrease in the time from when the ram started to approach the ewe until the completed mount. It is concluded that the responses of approaching the ewe, pawing the side of the ewe with chin resting on the ewe, and sniffing of the ewe (the latter occurring only during the training phase) are behavioral indicators that could be used for selection of easy-to-train rams for purposes of semen collection with an artificial vagina.

Isolation and characterization of spermatogenic cells from cattle, yak and cattleyak.

Cattleyak forms the first generation in the cross-breeding of cattle (Bos taurus) and yak (Bos grunniens), the purpose of which is to increase the yak's performance in meat and milk production. The female cattleyak is fertile while the male remains sterile due to spermatogenic arrest. The spermatogenic cells (including spermatogonia and spermatocytes) of cattle, yak and cattleyak have not been successfully isolated so far. In this work, spermatogenic cells were isolated from these bovid species with the STA-PUT method that has been previously used for germ cell sorting in human and mouse, and the isolated cells could be used to investigate the mechanisms involved in male sterility observed in cattleyak. The characteristics and size of the isolated cells were investigated through microscopic examination, and the cell types were identified by RT-PCR amplification of the marker genes. The purity of spermatogonia and spermatocytes isolated from each bovid species was found to be higher than 85%. The spermatogonium diameter of cattle (10.10 ±â€¯1.04 µm) and yak (14.90 ±â€¯2.30 µm) were significantly larger (P < 0.01) than that of cattleyak (8.60 ±â€¯0.92 µm). The spermatocyte diameter of cattle (19.40 ±â€¯1.50 µm) and yak (20.50 ±â€¯2.42 µm) were also significantly larger (P < 0.01) than that of cattleyak (17.70 ±â€¯2.05 µm). Therefore, the STA-PUT was again validated to be a convenient, economical and efficient method for isolation of spermatogenic cells as it yields more cells within a short time frame.

Mineral profiling of ostrich (Struthio camelus) seminal plasma and its relationship with semen traits and collection day.

Successful assisted reproduction techniques, with specific focus on in vitro semen storage for artificial insemination, are dependent on certain key elements which includes the biochemical profiling of semen. The objective of this study was to complete an ostrich seminal plasma (SP) evaluation by inductively coupled plasma mass spectrometry (ICP-MS) among seven males at different daily intervals (day 1, 3, 7, 11, 15, 19, 21, 23, 25, 26, 27, 28) for a period of 28 days during spring (August to September) for mineral profiling. The effect of collection day and male on sperm concentration, semen volume and seminal plasma volume, was explored as well as the relationships amongst these specific sperm traits and SP minerals. Variation amongst SP mineral concentrations, accounted for by the fixed effects of sperm concentration, semen volume, seminal plasma volume, collection day and male, ranged from 18% to 77%. Male had the largest effect on variation in SP minerals, namely: phosphorus (P), potassium (K), calcium (Ca), sodium (Na), boron (B), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), molybdenum (Mo), barium (Ba), arsenic (As) and selenium (Se). Sperm concentration instigated fluctuations of P, magnesium (Mg), B, zinc (Zn), Fe, aluminium (Al), Se, manganese (Mn) and lead (Pb). Semen volume had an effect on Na, K, B, Pb and Ba while seminal plasma volume only influenced variation in Na. There were fluctuations among collection days of specific micro minerals, Ni and Mo, with initial Ni concentrations being relatively greater and Mo at lesser concentrations. Semen volume, seminal plasma volume and sperm concentration varied amongst males. Sperm concentrations during the initial collection days, 1 and 3, were less than that for days 7 to 28. Significant variation of SP minerals and sperm characteristics among ejaculates and males suggest an association of these specific elements with sperm function and are, therefore, considered to be of potential importance to success of assisted reproduction technology for the ostrich. The relationship amongst sperm concentration and collection day confirms the need to conduct an initial period of collection to stabilise a greater sperm concentration to optimise sperm numbers for artificial insemination purposes.

Lunar and climatic effects on boar ejaculate traits.

There is evidence that phases of the moon affect wild animal behaviors including reproduction. There is, however, little evidence of moon phase effects on domestic livestock reproduction. This study investigated the effects of moon phase and climatic variables on boar ejaculate traits. Records of 4149 semen collections from boars of nine different breeds at one boar stud were used. The response variables were volume of ejaculate, concentration of sperm in the ejaculate, and number of doses obtained per ejaculate. Moon phase, greatest daily temperature (T), least daily T, average daily relative humidity (RH), temperature-humidity index (THI), season and the interaction of moon phase with season were analyzed at the day of collection and 45 days prior to date of collection as a proxy of initiation of spermatogenesis. For both dates analyzed season and the interaction of season with moon had significant effects (P < 0.05) on the volume of the ejaculate. Moon phase had a significant effect (P < 0.05) on volume of ejaculate at the day of collection. Sperm concentration was affected (P < 0.05) by the interaction of moon phase with season, high and low temperature, THI, RH and breed. Season had an effect (P < 0.01) on concentration of sperm at the initiation of spermatogenesis. For doses that could be used for AI that were obtained/ejaculate, there were effects of moon phase, season, the interaction between season and moon phase and breed (P < 0.05) at collection day and at the initiation of spermatogenesis. There was an interaction (P < 0.0001) between season and moon phase for volume of ejaculate, sperm concentration and number of doses obtained per ejaculate at date of collection and at day of initiation of spermatogenesis. The significant interaction of season and moon phase on boar semen traits suggests that to maximize productivity of modern swine production systems determining a collection schedule in some seasons relative to moon phase may be advantageous.

Production of functional sperm by subcutaneous auto-grafting of immature testes in rainbow trout.

Sexually mature individuals are indispensable for breeding programs. Salmonids require a long period before reaching sexual maturity, so we aimed to shorten the period required to obtain functional sperm by grafting immature testicular fragments into mature recipients, which we predicted would allow the grafted testicular fragments to skip the long pre-pubertal period. First, we demonstrated successful subcutaneous auto-grafting of testicular fragments in rainbow trout. Unilateral testectomy was performed, and the isolated immature testicular fragment was auto-grafted into the subcutaneous space along the back of recipient fish. The grafted testicular fragments developed synchronously with the recipients' testis remaining in its body cavity, and both eventually produced functional sperm. Next, immature testicular fragments were auto-grafted into the subcutaneous space of sexually mature males. We achieved this, without immune rejection, by isolating and cryopreserving testes from immature fish, and rearing these unilaterally testectomized fish until sexual maturity. The cryopreserved testes were then auto-grafted into the original, now spermiating fish. The grated immature testicular fragments differentiated and produced functional sperm within 5 months after grafting. By combining this grafting method with a technique to avoid immune rejection, we expect to develop a practical method for producing sperm in a shorter period in salmonids.

Effect of sperm selection techniques in frozen/thawed cat spermatozoa on sperm motility analyzed by CASA system / Efecto de técnicas de selección espermática en espermatozoides congelados/descongelados de gato sobre la motilidad espermática analizada mediante sistema CASA

SUMMARY: Freeze/thawing process reduces sperm survival and fertilizing ability of cat spermatozoa, with sperm motility being the most sensitive sperm parameter altered, due to cryo-damage. In this context, swim-up and density gradient processing methods can help to recover high motile and normal spermatozoa. Maximizing the use of frozen semen sample is essential, especially in endangered felids or high value cats in which sample size, number of samples or access to semen collection is reduced. To our knowledge, there is no previous report describing an in depth analysis of sperm motility improvement, after sperm selection techniques in frozen cat semen. Accordingly, we evaluated the effect of percoll gradient (PG) and swim up (SU) sperm selection techniques on sperm motility parameters and sperm recovery rate in frozen/thawed spermatozoa of domestic cat. Next, we evaluated the individual effect of the cat over sperm motility after PG sperm selection of frozen/thawed spermatozoa. SU and PG improved significantly all sperm motility parameters of frozen/thawed cat spermatozoa compared to simple washing. However, PG allows better sperm recovery from the original frozen sample and works mostly homogeneously among individual cats. This new information could help to maximize the use of frozen semen in endangered felids or high value domestic cats for its subsequent application on in vitro fertilization and artificial insemination.

RESUMEN: El proceso de congelación/descongelación reduce la sobrevivencia espermática y la habilidad para fertilizar en los espermatozoides de gato, siendo la motilidad espermática el parámetro más sensiblemente alterado debido al daño por frío. En este contexto, los métodos de procesamiento de swim-up y gradiente de densidad pueden ayudar a recuperar los espermatozoides normales y de alta motilidad. Maximizar el uso de una muestra de semen congelado es esencial, especialmente en felinos amenazados o en gatos de alto valor en los cuales el tamaño de muestra, número de muestras o el acceso a la colecta de semen son reducidos. Para nuestro conocimiento, no hay reportes previos que describan un análisis profundo del mejoramiento de la motilidad luego de técnicas de selección espermática en semen congelado de gato. De acuerdo a esto, evaluamos el efecto de las técnicas de selección espermática gradiente de percoll (PG) y swim up (SU) sobre los parámetros de motilidad y porcentaje de recuperación de espermatozoides congelados/descongelados de gato doméstico. Luego, evaluamos el efecto individual del gato sobre la motilidad espermática luego de la selección espermática con PG en espermatozoides congelados/descongelados. SU y PG mejoraron significativamente todos los parámetros de motilidad espermática de los espermatozoides congelados/descongelados comparado con el lavado simple. Sin embargo, PG permitió una mejor recuperación de espermatozoides desde la muestra congelada original y funcionó en su mayoría de manera homogénea entre los gatos individualmente. Esta nueva información puede ayudar a maximizar el uso del semen congelado en felinos amenazados o en gatos de alto valor para su posterior aplicación en fecundación in vitro e inseminación artificial.

Sperm subpopulations in ejaculated sperm and spermatozoa recovered from ovine epididymides up to 48h after death.

The objectives of this study were threefold: to identify subpopulations of sperm based on the kinetics of frozen/thawed sheep epididymal spermatozoa or semen collected with an artificial vagina; to evaluate the effects on sperm subpopulations in the thawed samples of post mortem storage at room temperature and the addition of 20% of seminal plasma to the freezing extender and to correlate the percentage of subpopulations with gestation rate following artificial intrauterine insemination. The categorization of the subpopulations was based on sperm kinetic data from Computer Assisted Sperm Analysis (CASA). A hundred ewes were inseminated with thawed spermatozoa and gestation rate was correlated with the proportions of each subpopulation using Pearson correlation matrix and linear regression. Three distinct subpopulations were identified in the thawed samples of either ovine ejaculate collected in artificial vaginas (AV) or ovine spermatozoa retrieved from the cauda epididymis. Subpopulation 1 (SP1) was characterized by spermatozoa with slow and non-linear motion, subpopulation 2 (SP2) was classified as hyperactived spermatozoa and subpopulation 3 (SP3) was composed of spermatozoa with fast, linear motion. The largest subpopulation in all groups was SP1. The semen collected in an artificial vagina had a higher (P<0.05) percentage of SP2 and lower (P<0.05) percentage of SP1 when compared to spermatozoa recovered after death. Increasing time of storage after death had a detrimental effect on sperm samples, increasing (P<0.05) the percentage of SP1 and decreasing (P<0.05) SP2. Length of storage after death was the only variable that influenced, with an inversely proportional relationship, SP3. In samples stored for 48h after death no SP3 spermatozoa were present. The addition of seminal plasma to the cryopreservative decreased (P<0.05) the subpopulation of hyperactived spermatozoa (SP2). We conclude that, after thawing there are three sperm subpopulations in the spermatozoa obtained from the cauda epididymides and the semen collected in AVs and that the relative proportions of these subpopulations varies with the time of storage post mortem and the presence of 20% of seminal plasma in the extender. However, we conclude that these subpopulations do not correlate with fertility after intrauterine artificial insemination.

Sperm evaluation of Jungle Cat (Felis chaus) obtained by urethral catheterization (CT) after medetomidine administration.

This study aimed to evaluate semen from Jungle Cat (Felis chaus) by urethral catheterization (CT) after medetomidine administration that offers feasible and different approaches to obtaining good quality sperm, especially in wild felids. Accordingly, this method was tested in five Jungle Cats. After general anesthesia with the α2-agonist medetomidine (which also stimulates semen release into the urethra) and ketamine, an abdomen ultrasound was performed to locate dilation of the first segment of the urethra (prostatic urethra). A commercial Tom cat urinary catheter 3-5 (depending on the size of the animal) was advanced into the urethra to reach the semen full dilated primary region of the urethra, so as to allow semen collection into the lumen of the catheter by capillary forces. After retraction, sperm volumes between 69 ± 27.92 yielded motility of 77.13 ± 14.15 (mean ± SD) with a mean sperm concentration of 75.13 ± 17.05 million/ml. The results of this study showed that semen collection in jungle cat is feasible, using this method. This study describes a simple, useful in field, inexpensive method which does not require the training of the animal and is better than other methods. Samples have normal pH, suitable color and consolidation, high concentration and lower contamination with excellent motility in Jungle Cat and potentially, other wild felid species, as an alternative to electro-ejaculation.

Effects of hormonal stimulation on the concentration and quality of excreted spermatozoa in the critically endangered Panamanian golden frog (Atelopus zeteki).

Knowledge of basic gamete biology is critical to better protect and propagate endangered amphibian species and also to develop reproductive technologies combined with germplasm cryopreservation. The objectives of the study were to test different hormonal stimulations and then characterize the quantity and quality of Panamanian golden frog (Atelopus zeteki) spermatozoa. Following intraperitoneal injection of the gonadotropin-releasing hormone agonist (des-Gly10, D-Ala6, Pro-NHEt9--GnRH 1, 2 or 4 µg/g of body weight), human chorionic gonadotropin (hCG; 5 or 10 IU/gbw), or Amphiplex™ (0.4 µg/gbw GnRH-A + 10 µg/gbw metoclopramide hydrochloride), spermic urine samples from 29 males were collected at different time points (from 0.5 to 24 h post-injection) to analyze the concentration, motility, and morphology of the spermatozoa. Peak of sperm concentration was observed at 3.5 h post injection for all hormonal treatments. Amphiplex™ led to the highest sperm concentrations (4.45 ± 0.07 × 106 cells/mL) followed by 4 µg/gbw GnRH-A (2.65 ± 0.21 × 106 cells/mL). Other stimulation protocols and doses induced sperm production, but at lower levels (ranging from 1.34 to 1.70 × 106 cells/mL). More than 60% of spermatozoa were motile following all treatments but the highest motility (>90%) was obtained from the 10 IU/gbw hCG treatment. Spermic urine samples obtained with all hormone treatments had higher pH (ranging from 7.1 to 7.8) than the urine alone (6.7-6.8). Spermatozoa were filiform and elongated with an apical acrosome, a mitochondrial sheath, a small midpiece and a long tail with an undulating membrane. More than 80% of cells were morphologically normal and 50-70% had intact DNA. These sperm characteristics were not influenced by hormonal treatments. This first comprehensive characterization of sperm samples following optimized hormonal stimulations in A. zeteki lays the foundation for more fundamental studies, reproductive technologies, and future preservation strategies.