Sperm Suspensions Research Articles

Broodstock development, captive breeding and seed production of bagrid catfish, Mahanadi rita, Rita chrysea (Day, 1877)

The present article describes about the broodstock development, induced breeding, and seed production of the Mahanadi rita, Rita chrysea, a medium-sized catfish, of the bagridae family under captive conditions. Juvenile fish collected from the Mahanadi river and raised to broodstock in the captivity and successfully breed in captive condition for the first time in India through hormonal induced breeding. Breeding performance of different broodstock size groups were evaluated to identify the suitable brooder size for successful captive breeding. Two broodstock size groups of female fish (n = 12, each group) i.e., 50–80 g and 90–130 g were selected. The female and male brood fish were induced with a synthetic hormone, Ovatide™ at 1 and 0.5 ml kg−1 body weight, respectively. All the hormone induced female brood fish were stripped for eggs 13–15 h of post-injection. The stripped eggs were fertilized with sperm suspension solution and incubated in glass jar for 24–26 h for hatching. The results revealed that the bigger sized broodstock fish group 90–130 g produced significantly higher (P < .01) number of eggs than the 50–80 g broodstock size. The total and relative fecundity of 90–130 g broodstock fish size were estimated to be 13,751 ± 619 and 12,123 ± 443, respectively. The egg quality evaluation parameters, such as fertilization rate (75.32 ± 0.71%) and hatching rate (63.88 ± 1.09%) were also significantly higher (P < .01) in the 90–130 g size brood fish. The bigger sized broodstock (90–130 g) group produced significantly higher sized eggs 1.37 ± 0.05 mm and hatchlings 4.3 ± 0.2 mm and 1.2 ± 0.3 mg compared with smaller broodstock group (50–80 g). Mahanadi rita seed production techniques were standardized through larval stocking density experiment. The larvae were stocked at different density levels to ascertain the optimum stocking density for mass scale seed production of the species in the indoor conditions. The 6-day-old larvae were stocked at different densities level, such as 2, 3, 4 and 6 numbers per litre of water. The study confirmed that 2 larvae L−1 stocking density group produced larvae with a significantly (P < .05) higher body weight gain 153.33 ± 1.88 mg and both 2 and 3 larvae L−1 density group showed significantly higher (P < .01) larval survival rate, 93.33% and 86.67 ± 2.23%, respectively.

Impact of post-thaw supplementation of semen extender with antioxidants on the quality and function variables of stallion spermatozoa

During cryopreservation procedures, the spermatozoa are exposed to physical and chemical stressors that generate an increase in the intracellular concentration of reactive oxygen species (ROS). If ROS concentrations are too great, this can lead to a state of oxidative stress that are detrimental to sperm quality. The aim of this study was to ascertain the profile the ROS production and assess the effects of post-thaw supplementation of a semen extender with different antioxidant compounds on the quality and function variables of frozen-thawed stallion spermatozoa incubated in vitro. Frozen-thawed stallion spermatozoa (2 × 106 cells/mL) were incubated with three different antioxidants (MnTBAP, NAC and FeTPPS) for 4 h at 38 °C. An untreated sperm suspension and a fresh sample were included as controls. Plasma membrane integrity (SYBR-14/PI), intracellular ROS concentration (DHE and ROS-ID™ total ROS/Superoxide Detection Kit), lipid peroxidation (BODIPY), DNA damage (TUNEL) and mitochondrial membrane potential (ΔΨm; TMRE/SYTOX) were evaluated by flow cytometry and fluorescence microscopy. In addition, sperm motility was evaluated using the ISAS system. Evaluations were performed at 0 and 4 h of incubation. The results indicate that superoxide anion is the main ROS produced by frozen-thawed stallion spermatozoa and that the use of MnTBAP improved sperm motility and viability, decreased the lipid peroxidation and DNA damage. In conclusion, this study provides relevant data to improve in vitro incubations conditions and to establish futures therapies using MnTBAP after thawing with the aim being to overcome the deleterious effects of semen cryopreservation and consequently preserve the stallion sperm quality through avoiding oxidative stress.

105 Functionality evaluation of two extenders for Leopardus geoffroyi sperm cryopreservation by interspecific IVF with domestic cat oocytes

Even though knowledge in sperm cryopreservation of endangered felids advanced in recent years, very little is known about suitable protocols to cryopreserve sperm from Leopardus geoffroyi (LG). In the present study, sperm obtained by electroejaculation from 5 different males were cryopreserved in either a Tes-Tris- or a lactose-based diluent (Gañan et al. 2009 Theriogenology 72, 341-352) with modifications in the freezing process using a one-step method: straws were placed horizontally on a metal rack, 4cm above the surface of liquid nitrogen in a styrofoam box, and kept for 10min before plunging them in LN. The objectives were to (1) compare in vitro motility and acrosome status of LG sperm cryopreserved in both extenders and (2) test functionality of LG sperm cryopreserved in both extenders through their ability to fertilize mature domestic cat oocytes. Straws were thawed by exposing them to air for 10s and then immersing them in a water bath at 37°C for 30s. The contents of the straws were poured into a sterile 1.5-mL microtube prewarmed to 37°C. The sperm suspension was diluted (1:3 vol/vol) by the slow (drop by drop) addition of a modified Tyrode’s solution. Sperm parameters, percentage of motile spermatozoa, and quality of motility were assessed and sperm motility index (SMI) was calculated as follows: [% motile sperm+(quality×20)]/2. Acrosome integrity was assessed by staining with Coomassie brilliant blue. For IVF, in vitro-matured domestic cat oocytes (n=238 Tes-Tris, n=239 lactose) were co-incubated with 0.5×105 motile spermatozoa/mL under 5% CO2 in air at 38.5°C for 18-20h (Pope et al. 2006 Methods Mol. Biol. 254, 227-244). Presumptive zygotes were cultured in vitro in 50-µL drops of modified Tyrode’s medium at 38.5°C in 5% CO2, 5% O2, 90% N2 atmosphere. Cleavage was assessed 48h postfertilization, and 5% FBS was added at Day 5 of in vitro culture. Blastocyst stage was evaluated at Day 8. Data was analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc., San Diego, CA, USA), significant at P&amp;lt;0.05. Results, mean (±standard error of the means), showed that SMI and acrosome integrity (pre- and post-thawing) were similar for both extenders: prethawed (SMI=56±3.3v. 59±5.5; acrosome integrity=88±3.0% v. 90±2.0%), and post-thawed (SMI=46±5.0v. 44±7.0; acrosome integrity=57±7.5% v. 68±2.4%) Tes-Tris v. lactose, respectively. For IVF, results showed a high cleavage rate in both groups (117/238, 49% v. 117/239, 49%), and a high development to morula (96/238, 40% v. 94/239, 39%) and to the blastocyst stage (61/238, 26% v. 51/239, 21%) for all males Tes-Tris v. lactose, respectively. There were no significant differences between groups at any development stage. In conclusion, we found that both extenders can be used to cryopreserve LG sperm maintaining functional conditions and that fertilizing capacity can be tested using in vitro-matured domestic cat oocytes.

Allurin and egg jelly coat impact on in-vitro fertilization success of endangered Albanian water frog, Pelophylax shqipericus

Amphibian egg-jelly coat plays an important role in successful fertilization and development. Here, we ask whether proteins like allurin in the jelly coats of frog eggs might influence fertilization rate success. Using in vitro fertilization of Albanian water frog, Pelophylax shqipericus, we found that body cavity eggs or eggs deprived of jelly coat were not fertilized, compromising the success of in vitro fertilization procedure. When de-jellied eggs were inseminated with sperm suspension, the fertilization efficiency is dramatically decreased even inhibited, suggesting that the gel structure is one of the major factors in the achievement of fertilization in the frogs. Fertilization of de-jellied eggs with sperm pre-treated with egg jelly coat, restored the fertilization competency. Such a result suggests that egg jelly coat probably guides the sperm to the egg surface while maintaining the fertilization ability, contributing to a successful in vitro fertilization of Pelophylax shqipericus.

Evaluating the impact of moulting and chilled storage of spermatophores on the integrity of plasma membrane, acrosome and DNA of black tiger prawn ( Penaeus monodon ) spermatozoa

To explore the impact of moulting and short-term chilled storage of spermatophores on the sperm quality for a commercially important penaeid prawn, spermatozoa of Penaeus monodon from early (B-C), middle (D0-1) and late (D2-3) moult stages were compared for sperm quality parameters relating to the structural integrity of plasma membrane (Viab%), acrosome (AR%) and DNA (SDF%) after being stored at 4°C for 0–26 days. The three different sperm extenders used for chilled storage included artificial lobster haemolymph (AH), calcium free saline (CS) and filtered seawater (FS); two storage conditions were applied either as a free sperm suspension or retained within the intact spermatophore. Results showed that (a) the lowest natural AR% was shown for B-C spermatozoa in CS whereas the highest levels were for B-C, D0-1 and D2-3 spermatozoa in FS and D2-3 spermatozoa in AH; (b) the calcium ionophore A23187 agent used in this study was able to increase the mean AR% by 6.22%; (c) the Viab% was significantly lower in CS than that in FS; (d) the SDF% significantly increased over the period of chilled storage for B-C and D0-1 spermatozoa, while the SDF% of D2-3 spermatozoa was initially elevated and did not change significantly over time; and (e) there was no difference in sperm quality between two storage conditions. This study has successfully demonstrated the moult-related variance in the percentages of acrosome-reacted and DNA-damaged spermatozoa, providing evidence of moult-related spermatophore renewal cycle in this species.

Removal of seminal plasma prior to liquid storage of boar spermatozoa: A practice that can improve their fertilizing ability

Seminal plasma (SP) plays a vital role in the maintenance of sperm function and integrity along with being involved in their communication with the female reproductive tract. Under in vitro conditions, although it is generally accepted that boar semen is better preserved when SP is diluted (extended) or removed (cryopreserved), its role during storage is not completely elucidated. In this context, the current study sought to determine the role of SP during storage of boar spermatozoa at 17 °C for 72 h. Thus, two treatments were prepared with semen from the sperm-rich fraction (SRF) of boar ejaculate previous to storage in liquid state: 1) PSP: semen directly extended in Beltsville Thawing Solution (BTS), and 2) ASP: semen first centrifuged with subsequent removal of supernatant (containing SP) followed by suspension of sperm in BTS. From this, two experiments were conducted separately in this work: 1) in vitro and 2) in vivo assays. The former aimed to evaluate how sperm capacity responds to in vitro capacitation (IVC) and whether their quality is affected by previous exposure to SP. In the latter, the objective was to understand how important these previous conditions can be for reproductive performance after artificial insemination (AI). According to our results, the previous removal of SP does not affect sperm quality and the response of these cells to IVC (P > 0.05) along with resulting in a lower percentage of acrosome damage in them [12.87 ± 0.76 (ASP) vs. 16.38 ± 0.73 (PSP)] (P < 0.05). This improved preservation of acrosome integrity in the absence of SP can explain the higher fertility rate (%) [63.27 ± 23.47 (ASP) vs. 38.57 ± 16.30 (PSP)] and number of implanted embryos at 28 days after AI (13.71 ± 4.88 (ASP) vs. 7.16 ± 4.02 (PSP)] (P < 0.05) presented by gilts inseminated with seminal doses of ASP. In conclusion, removal of SP prior to liquid storage of boar sperm for 72 h can be beneficial for their fertilizing ability.

Dose and time-dependent effects of sodium fluoride on sperm motility: An in vitro study.

The present study was undertaken to investigate the toxic effects of sodium fluoride (NaF) on sperm motility and abnormality. Treatment of epididymal sperm suspensions with different doses of NaF (0.1, 1, 10, and 100 mg/ml) significantly reduced sperm motility at 15- and 30-min time intervals. Further, incubation at the same concentrations of NaF for 5-, 10-, and 15-min time intervals decreased sperm motility in a dose- and time-dependent manner. The present investigation revealed for the first time the adverse effect of lower doses of NaF (0.1 mg/ml) on sperm motility. Further, higher doses of NaF (10 and 100 mg/ml) caused a complete loss of sperm motility with a concomitant increase in sperm abnormality, which reflected the toxic effect of NaF on spermatozoa and its capability in fertilization.

Intra-ooplasmic injection of a multiple number of sperm to induce androgenesis and polyploidy in the dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae)

SummaryPolyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml -1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.

Positive effect of progesterone on motility and velocity of fresh, vitrified without permeable cryoprotectants and frozen with permeable cryoprotectants human spermatozoa.

Steroid hormone progesterone has been found to play an important role in the migration of spermatozoa through the reproductive tract, as well as to induce hyperactive motility and increase sperm velocity. The aim of this study was to examine whether progesterone could induce beneficial effects in vitrified and slow-frozen spermatozoa. During the research process, 50 semen samples were divided into three treatment groups; noncryopreserved, slow-freezing and vitrification. After thawing and an incubation period of 2hr to induce capacitation, semen samples from each treatment group were treated with 50nM, 25nM progesterone and a control solution for 30min. Thereafter, the sperm suspensions were examined manually to assess the proportion of viable and motile spermatozoa, as well as using the CASA to evaluate the velocity parameters. The results indicated a higher proportion of progressively motile spermatozoa in vitrified teratozoospermic samples and improved velocity parameters in slow-frozen normozoospermic and teratozoospermic samples. The main conclusion of this research was that the used progesterone concentration of 50nM was sufficient to significantly improve the motility of vitrified teratozoospermic samples and velocity parameters of cryopreserved sperm samples. The present findings might have important implications in determining ways of improving the current low rates of motility in cryopreserved spermatozoa.

Concentrations of non-permeable cryoprotectants and equilibration temperatures are key factors for stallion sperm vitrification success

Vitrification is based on rapid freezing by direct exposure of sperm to liquid nitrogen (LN2). This study evaluated the effect of non-permeable CPAs and equilibration temperature on stallion sperm quality after vitrification. In Experiment 1, different concentrations of sucrose (20, 50, 100 mM; mmol/L) and bovine serum albumin (BSA 1%, 5%, 10%) were compared including different temperatures for the equilibration (≈22 °C or 5 °C). Vitrification was performed dropping 30 μl sperm suspension directly into LN2. In Experiment 2, conventional sperm freezing using 2.2% of glycerol in 0.5 ml straws, frozen in LN2 vapours, was compared to the sucrose and BSA extenders (and its combination) producing the most desirable results. Sperm motility, plasma membrane and acrosome integrity were statistically compared between treatments. Vitrification after sperm cooling at 5 °C with sucrose 20 mM (S20) or BSA 1% (BSA1) resulted in the greatest values (mean ± SEM) for most of the sperm variables assessed. With use of the combination (S20 + BSA1/5 °C), there were greater values (P<0.001) than freezing with glycerol for total (55.67 ± 2.99 vs 35.41 ± 2.96) and progressive sperm motility (38.32 ± 3.05 vs 14.42 ± 1.80), plasma membrane integrity (66.61 ± 2.69 vs 49.16 ± 2.60), intact-acrosomes (49.19 ± 2.60 vs 14.91 ± 1.57) and most of the kinetics assessed, respectively. In conclusion, stallion sperm can be vitrified after cooling at 5 °C using a combination of 20 mM sucrose and 1% BSA based extender and this is a promising alternative compared with conventional sperm freezing using glycerol.

Assessment of azadirachtin-A, a neem tetranortritarpinoid, on rat spermatozoa during in vitro capacitation.

The exploration of the biological assessment of technical azadirachtin, a tetranortritarpinoid from the neem seed kernel, was reviewed. The present study was, therefore, designed to evaluate the dose-dependent in vitro effects of azadirachtin-A, particularly on the functional studies and determination of molecular events, which are critical in the process of sperm capacitation. To assess the effects of the azadirachtin-A on the functional studies, sperm capacitation, the total sperm adenosine triphosphate levels, acrosome reaction (AR), the sperm-egg interaction and the determination of molecular events like cyclic adenosine-3',5'-monophosphate and calcium levels, the appropriate volumes of the sperm suspension were added to the medium to a final concentration of 1×106 sperm/mL and incubated in a humidified atmosphere of 5% CO2 in air at 37°C. The increasing quantities 0.5-2.0 mM/mL and the equivalent volumes of 50% dimethyl sulfoxide were added to the control dishes prior to the addition of spermatozoa and then observed at various time-points for motility and other analyses. Results revealed the dose- and time-dependent decrease in the functional consequence of capacitation, i.e. the percentage of motile spermatozoa, motility score and sperm motility index, levels of molecular events in spermatozoa, followed by declined spontaneous AR leading to lesser binding of the cauda epididymal sperm to the Zona pellucida. The findings confirm the inhibition of rat sperm motility by blocking some biochemical pathways like energy utilization. They also demonstrate that sperm capacitation is associated with the decrease in AR and that the levels of molecular events in spermatozoa can guide us towards the development of a new male contraceptive constituent.

Optimalisasi Pembekuan Sperma Limbah Kauda Epididimis Kambing Lokal dengan Metode Bertahap dan Stabilisasi

Buck slaugthering produce waste such as testicles including epididymis which contain fertile sperm. Utilization of cauda epididymis as the sources of sperm for producing goat frozen sperm was not reported yet. The aims of this study were improving the frozen-thawed sperm using stabilization and multistep methods which recovered from the waste of buck slaughtering as the source of sperma. Cauda epididymis spermatozoa which was washed then diluted using extender 1 (Tris-citrate-antibiotics) and extender 2 (extender 1- glycerol-egg yolk). The extender 2 addition was performed by single or multistep methods then freezed. Modification in the pre freezing proces were performed by comparing the conventional equilibration and stabilization methods. The sperm suspension was incubated in 4°C for 2 hours after filling-sealing into straws on the equilibration group whether the stabilization group was cooled in tube 15 mL. All cooled straws from both groups were placed 4 cm horizontally on liquid nitrogen surface for 10 minutes and then plunged into liquid nitrogen for storage. The evaluation of motility parameters such as pattern of the movement and motility percentation were done followed the standard methodology. The student t-test, correlation and one-way ANOVA were used for data analysis with P&lt;0.05. The results showed that multistep dilution method could increase the motility (25.0 ± 1.8 %) compared with single step (18.3 ± 1.7 %). Pre freezing method with stabilization also resulted higher motility (24.2 ± 2.0 %) than equilibration method (17.5 ± 2.8 %). The pattern of the movement were not different between all methods and its combination. The multistep dilution method and stabilization cooling method as well as its combination seems could increase the quality of frozen-thawed cauda epididymis spermatpzoa of local buck.

The Usefulness of Sperm Kinematics in Drug-Induced Toxicity Assessment.

In vitro drug-induced toxicity assessment demands the usability and validation of suitable cell models, obtained non-invasively and pain-free. Sperm suspensions, painlessly and easily obtained from breeding boars, have been proven as suitable biosensors for pre-clinical toxicology screening and ranking of lead compounds in the drug development processes on a kinematics-based assay. Having a limited number of mitochondria and depending on these mitochondria and on cytoplasmic glycolysis for the energy needed for motility and for plasma membrane functionality, spermatozoa become a suitable model for capturing multiple modes of action of drugs and other chemicals acting via measurements of sperm motility. In this MiniReview, the usability of boar spermatozoa as detectors of cytotoxicity based on sperm motility measurements is discussed.

From flagellar undulations to collective motion: predicting the dynamics of sperm suspensions.

Swimming cells and microorganisms are as diverse in their collective dynamics as they are in their individual shapes and propulsion mechanisms. Even for sperm cells, which have a stereotyped shape consisting of a cell body connected to a flexible flagellum, a wide range of collective dynamics is observed spanning from the formation of tightly packed groups to the display of larger-scale, turbulence-like motion. Using a detailed mathematical model that resolves flagellum dynamics, we perform simulations of sperm suspensions containing up to 1000 cells and explore the connection between individual and collective dynamics. We find that depending on the level of variation in individual dynamics from one swimmer to another, the sperm exhibit either a strong tendency to aggregate, or the suspension exhibits large-scale swirling. Hydrodynamic interactions govern the formation and evolution of both states. In addition, a quantitative analysis of the states reveals that the flows generated at the time scale of flagellum undulations contribute significantly to the overall energy in the surrounding fluid, highlighting the importance of resolving these flows.

171 Effects of Combination of Korean Red Ginseng Extract and Hydrogen-rich Water on Spermatogenesis and Sperm Motility in the Mouse Model

Male fertility was majorly declined with aging due to age-related oxidative stress. Natural therapies with herbal supplements have been increasing popularly as an option to treat male sub- or infertility. Hydrogen rich water (HRW) is a potent antioxidant and Korean red ginseng (KRG) has a various function including the improvement of spermatogenesis and sperm motility. Therefore, this study is aimed to investigate the effect of combination of hydrogen-rich red ginseng water (HRGW) on spermatogenesis and sperm motility in the mice according to the age. C57BL/6 male mice of young (3 months) and aged group (12 months) were randomly assigned to four groups and then they were administered once daily in a volume of 10mL/kg of body weight by an oral zoned needle for a period of 4 weeks: (1) Control group (n=10), (2) HRW treatment group (n=10), (3) KRG treatment group (n=10, 50mg/kg/day); (4) HRGW treatment group (n=10). After treatment, sperm count and motility were measured using sperm suspension released from cauda epididymis, and serum FSH, LH, testosterone and reactive oxygen species (ROS) were measured. Testicular morphology was examined by histological H&E staining. Expression of anti-oxidation (PPx3, PPx4, GSTm5 and GPx4), spermatogenesis (inhibin-α, Neptin-4, and CREM), antiaging (Sirt1 and Sirt2), and angiogenesis (visfatin and VEGF)-related genes in the testes were examined with Real-time polymerase chain reaction (real-time PCR).

Development of sea bream (Sparus aurata) semen vitrification protocols

The long-term goal of this research project is to set up efficient protocol that can be used to develop a standardized approach for vitrification of marine fish spermatozoa. In particular, the aim of the present study was to develop a vitrification protocol for sea bream (Sparus aurata) spermatozoa. To draw up the protocol, we tested two different dilution media (1% NaCl and Mounib medium), three different vitrification devices (loops, drops and cut straws), different cryoprotectants (CPs) and three different equilibration times (30, 60 and 120 s). The effect of the different vitrification procedures on spermatozoa quality was checked by measuring spermatozoa motility rate and viability, mitochondrial membrane potential and the fertilizing ability of both fresh and post-thawed gametes.The best result was obtained by dropping directly into liquid nitrogen 20 μl of spermatozoa suspension (drop-wise method) diluted with Mounib buffer containing 10% Me2SO + 10% glycerol. The addition of a mixture of anti-freezing proteins, AFPI and AFPIII, to Mounib buffer significantly increases the spermatozoa quality following vitrification so confirming the usefulness of AFPs in improving the quality of gametes subjected to the vitrification process. The present study proves that vitrification offers an alternative to conventional sperm cryopreservation also in this species.

130 In Vitro Production of Hybrid Desert Bighorn×Domestic Sheep Embryos Using Frozen–Thawed Epididymal Semen from a Hunter-Harvested Ram

Although considered a species of least concern by the International Union for Conservation of Nature red list, the Desert Bighorn sheep (Ovis canadensis nelsoni) is listed in Appendix II of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) in Mexico, due to population size and the lack of protected areas. Postmortem epididymal sperm collected from a hunter-harvested Desert Bighorn sheep ram in Mexico, with an unofficial Safari Club International score of 197 2/8 and an estimated 5.5 years old, were used to evaluate the in vitro production (IVP) of embryos using postmortem-collected ram sperm. Testicles with epididymides were placed in the refrigerator ~45 min after harvest. Sperm were extracted from each epididymis and assessed separately for total motility (TM), progressive motility (PM), and membrane integrity using a phase contrast microscope. The sperm suspension was obtained from the distal end of both epididymides and cryopreserved 12 h postmortem using triladyl with egg yolk. Membrane integrity and morphology were evaluated using Eosin-Nigrosin stain. Sperm DNA fragmentation was analysed using the Halomax kit (Halosperm SL, Madrid, Spain) with fluorescence microscopy. Centrifugation with density gradient PureSperm (Nidacon International, Mölndal, Sweden) was used to remove dead sperm and debris before IVF. Ovaries were collected from Domestic sheep (Ovis aries) at a local slaughterhouse. The maturation medium was TCM-199 with Earle’s salts and a modified Tris-buffered medium was used for fertilization. Frozen straws of sperm from the Desert Bighorn ram were thawed for 45 s at 37°C. Sperm were diluted with modified Tween medium B with milk powder (mTBM) to a final concentration of 5 × 106 cells mL−1. The gametes were co-incubated for 18 h under previously described conditions. The cumulus cells were mechanically removed from zygotes and grown using a co-culture with granulosa cells in sequential media SOF1-SOF2. With regard to sperm collection, epididymis 1 produced 29 straws of sperm (0.25 mL, 136 × 106 sperm mL−1) and epididymis 2 produced 32 straws of sperm (0.25 mL, 68 × 106 sperm mL−1). The sperm sample used for IVF had TM of 60% and PM of 30%. Live dead staining of fresh sperm showed 68% live (i.e. intact cell membranes) and 28% post-thaw. Regarding DNA integrity, only 2% of sperm had DNA fragmentation at 0 h. Of 15 Grade 1 oocytes used for IVF, 4 cleaved (27%), with 1 developing to blastocyst stage (25%). The results show that frozen–thawed epididymal sperm collected from a recently deceased Desert Bighorn ram can provide a valuable source of sperm for IVP of embryos. These results also provide new information on Desert Bighorn sheep reproductive parameters for use in health assessment, or reproduction and conservation management through gene banking and assisted reproductive techniques.

98 Assessing Endangered Felid Puma concolor Sperm Fertility by In Vitro Fertilization with Domestic Cat Oocytes

The Puma concolor population has been decreasing during the last 30 years. Semen cryopreservation of this species has been accomplished successfully and offers the possibility of preserving endangered species. We previously showed that fertilizing capability of wild felid spermatozoa can be evaluated using intracytoplasmic sperm injection (ICSI) with in vitro-matured domestic cat oocytes (Moro et al. 2014 Reprod. Domest. Anim. 49, 693-700). Due to the lack of homologous oocytes, we evaluated the capability of the Puma concolor sperm to induce domestic cat oocyte fertilization and subsequent pre-implantation embryo development. In the present study, cryopreserved sperm obtained by electroejaculation from five different males were used for IVF of in vitro-matured (IVM) domestic cat oocytes. Straws were thawed by exposing them to air for 10 s and then immersing in a 37°C water bath for 30 s. The contents of the straws were poured into a sterile 1.5-mL microtube pre-warmed to 37°C. The sperm suspension was diluted (1:3 v/v) by the slow (drop-by-drop) addition of a modified Tyrode’s solution. For IVF, IVM oocytes (n = 370) were co-incubated with 0.5 × 105 motile spermatozoa mL−1 in an atmosphere of 21% O2 in air at 38.5°C for 18 to 20 h. Presumptive zygotes were cultured in vitro in 50-μL drops of modified Tyrode’s medium on 6.5% CO2 in air at 38.5°C. Cleavage was determined at 48 h post-fertilization, and 5% FBS was added at Day 5 of in vitro culture. Blastocyst stage was evaluated at Day 8. Results (mean ± SEM) showed a high cleavage rate (179/370, 49.0 ± 4.0%), and a high development to morula stage (137/370, 34.4 ± 7.2%), and to blastocyst stage (94/370, 23.4 ± 4.7%) for all males. These results indicated that Puma concolor spermatozoa can induce domestic cat oocyte activation and development to blastocyst stage in similar rates to domestic cat homologous IVF: IVM oocytes (n = 291), cleavage rate (199/291, 67.1 ± 6.1%), development to morula stage (144/291, 47.8 ± 4.9%), and to blastocyst stage (86/291, 30.1 ± 1.6%). In conclusion, we demonstrated that domestic cat oocyte can be used to evaluated cryopreserve sperm samples from another felid species.

Vitrification of dog spermatozoa: Effects of two cryoprotectants (sucrose or trehalose) and two warming procedures

Sperm vitrification is a low cost and simple technique that does not require special equipment and may represent an attractive alternative to the costly and time consuming conventional dog spermatozoa cryopreservation techniques. The objective of this study was to evaluate different cryoprotectants and warming temperatures on the vitrification of dog spermatozoa. Pooled semen samples from 10 beagle dogs were vitrified with four extenders, based on Tris, citric acid and glucose, 20% egg yolk (TCG-20% EY) and different combinations of sucrose and/or trehalose: 250 mM sucrose; 250 mM trehalose; 125 mM sucrose + 125 mM trehalose; 250 mM sucrose + 250 mM trehalose. Samples were vitrified by dropping 50 μL of sperm suspension directly into liquid nitrogen. After vitrification, warming was done either fast (at 65 °C for 2–5 s) or slow (at 37 °C for one minute). Motility was assayed using a computer-aided sperm analysis (CASA) system; membrane integrity and acrosomal status were analyzed by fluorescence microscopy. For comparison, samples were also conventionally frozen in liquid nitrogen vapor using a TCG-20% egg yolk extender plus 5% glycerol. Frozen straws were thawed in a water bath at 37 °C for 30 s. Poorer motility results (P < 0.05) but similar viability were obtained when vitrification was performed, compared to conventional freezing (P > 0.05). When vitrification was used, cryoprotectants containing either 250 mM sucrose or 250 mM trehalose and warmed at 37 °C returned the best sperm quality variables.

Generation of viable progeny from dead brooders of endangered catfish Clarias magur (Hamilton, 1822)

The obligatory air-breathing catfish Clarias magur is a prime candidate for aquaculture owing to its unique taste, high growth rate, and hardy nature. However, recently the IUCN has listed the species under the endangered category because the population has critically declined in the wild. The sexually mature C. magur brooders are often collected from their natural habitats for seed production in captivity. In many cases, the brooder dies due to handling injuries or confinement stress. In this study, we demonstrated that viable progeny could be generated from freshly dead sexually mature C. magur. Three hours after death, the gonads were excised, macroscopically examined and gamete viability was evaluated. Artificial fertilization was performed by mixing the sperm suspension with the eggs. Water was added after 1 min of mixing to activate the fertilization process. We observed 85%-93% fertilization success from gametes derived from dead donors as opposed to 90%-95% from those derived from live control donors. The embryos showed normal development and resulted in the generation of 88%-92% viable progeny, which was similar to the progeny derived from control donors (92%-93%). The results obtained in this study will have profound implications in enhancing the seed production of endangered C. magur and could potentially be applied to other key commercially or endangered fish species.