The Effect of PGF2α Injection on Post-Thaw Motility in Sperm of Nubian Goats

This study aims to determine the effect of PGF2α injection on the post-thaw motility (PTM) in sperm of Nubian goats. Three male Nubian goats (3-4 years) with good reproductive ability were used. This study used a 3 x 3 Latin square design. The experimental animals received a physiological NaCl injection as a control (P1); 37.5 mg of PGF2α (P2), and 75 mg of PGF2α (P3). Semen was collected using an artificial vagina with one-week storage intervals between treatments. The collected semen was then diluted and frozen using a simple freezing method. Observation of semen quality before freezing included macroscopic and microscopic examinations. Macroscopic examination consisted of volume, pH, color, odor, and consistency, while microscopic examination consisted of motility, concentration, viability, and abnormality. PTM examination was done by mixing a drop of sperm suspension and one drop of physiological NaCl on an object glass and covered prior to observation under microscope.. The results were analyzed using a Latin square pattern variant analysis, followed by Duncan's test. The PTM values of sperm (%) of Nubian goats in P1, P2, and P3 respectively are 28.71±10.24, 50.03±13.70, and 54.84±12.04 (P<0.05). Injection of PGF2α to Nubian goats by injection increased the PTM.

Computer Assisted Sperm Analysis: A Review

Background: A semen analysis exam is a routine check that is done to evaluate fertility. The World Health Organization (WHO) recommended a manual method to obtain objective and standardized values. However, sometimes some errors can be found with this method such as motility. Computer-Assisted Sperm Analysis offers a way to reduce inaccuracies that often occur with manual methods. Reviews: CASA systems consist of a microscope which connected into a camera to detect microscopic sperm suspension images and a computer installed with special software to extract desired information and produce the desired output. In the morphological examination, CASA can reduce the coefficient of variation (CV) which is around 4.8% compared to the manual examination but the time required is longer than manual. CASA can visualize and evaluate sperm kinematics. Various parameters such as mean path velocity (VAP), curved velocity (VCL), straight-line velocity (VSL), lateral head displacement amplitude (ALH), or beat cross frequency can be obtained, and this allows a detailed view into the behavior of individual sperm. The limitations affecting CASA's ability to provide accurate results for sperm concentration and percentages of motile or progressively motile spermatozoa. Summary: CASA has several advantages through its ability to calculate more detailed parameters, but a qualified operator must operate it because there is some potential for misinterpretation. The combination of The Manual and CASA is highly recommended for better results.

Vitrification of Donkey Sperm: Is It Better Using Permeable Cryoprotectants?

Vitrification by direct exposure of sperm to liquid nitrogen is increasing in popularity as an alternative to conventional freezing. In this study, the effect of permeable cryoprotectant agents for donkey sperm vitrification was compared to an extender containing non-permeable cryoprotectants. First, three different concentrations of sucrose (0.1, 0.2, and 0.3 molar, M) and bovine serum albumin, BSA (1, 5, and 10%) were compared. Secondly, the concentration of non-permeable agents producing the most desirable results was compared to an extender containing glycerol as permeable agent. Vitrification was performed by dropping 30 μL of sperm suspension directly into LN2 and warming at 42 °C. Sperm motility (total, TM; and progressive, PM) and plasma membrane integrity, PMI (mean ± SEM) were statistically compared between treatments. Sucrose 0.1 M showed a significantly higher percentage of total sperm motility (21.67 ± 9.22%) than sucrose 0.2 M (14.16 ± 4.50%) and 0.3 M (8.58 ± 6.22%); and no differences were found in comparison to the control (19.71 ± 10.16%). Vitrification with sucrose 0.1 M or BSA 5% obtained similar results for TM (21.67 ± 9.22% vs. 19.93 ± 9.93%), PM (13.42 ± 6.85% vs. 12.54 ± 6.37%) and PMI (40.90 ± 13.51% vs. 37.09 ± 14.28); but both showed higher percentages than glycerol (TM = 9.71 ± 4.19%; PM = 5.47 ± 3.17%; PMI = 28.48 ± 15.55%). In conclusion, donkey sperm vitrification in spheres using non-permeable cryoprotectants exhibited better sperm motility and viability parameters after warming than sperm vitrification using extenders containing permeable cryoprotectants.

Methods for improving the functional and metabolic status of spermatozoa with a low-intensity laser in vitro

A method for improving the functional and metabolic status of spermatozoa, including increased motion and physiological activity of spermatozoa obtained from the seminal fluid of a healthy human in vitro and including the red wavelength radiation of a low-intensity laser, is characterized in that spermatozoa isolated from a healthy donor are washed with saline twice by centrifugation for 10 minutes at 1500 rpm and the sperm suspension is impacted on with a 632 nm semiconductor laser in the mode of variable pulse generation, with the pulse width of 2 ns, the frequency of 100 Hz, the radiant energy density of 0.56 j/cm2 , the exposure time of 1 minute, expressed in increased motion and physiological activity of spermatozoa.

41 Effect of vitrification on DNA integrity of human spermatozoa

The aim of our study was to investigate the effect of vitrification with sucrose on swim-up-prepared human spermatozoa in comparison with standard, conventional manual freezing with permeable cryoprotectants. After informed consent, 35 ejaculates were obtained from 35 patients with normozoospermia who were patients of a fertility clinic. All specimens used for this study had fulfilled the following quality criteria for spermatozoa concentration and motility on IVOS (Hamilton Thorne). Semen analysis was performed according to published guidelines of the World Health Organization (WHO Laboratory Manual for the Examination and Processing of Human Semen, 2010). After swim-up, each sample was centrifuged, resuspended with the basic medium (human tubal fluid + 1% human serum albumin) to achieve a concentration of 5×106 spermatozoa/mL, and finally aliquoted into two equal subsamples. Each of these aliquots was assigned to one of two groups: group 1 included conventionally cryopreserved spermatozoa and group 2 included spermatozoa that were vitrified. For conventional cryopreservation, freezing media (15% (vol/vol) glycerol, 20% (vol/vol) egg yolk) and citrate was added to the washed spermatozoa in a 1:2 ratio. The sperm suspension was aspirated into 0.5-mL straws (CryoBioSystem). Subsequent to the room-temperature incubation for 10min, straws were placed horizontally in the vapour phase for 15min and then submerged into liquid nitrogen. For thawing, cryopreserved straws were immersed in water (23°C) for 5min. For preparation of vitrification solution, the basic medium (human tubal fluid + 1% human serum albumin) was diluted 1:1 with 0.5M sucrose. Immediately after processing, the sperm suspension was diluted in a 1:1 ratio with the vitrification solution to reach a final sucrose concentration of 0.25M. The vitrification and sperm solution (300μL) were aspirated into the straws 0.5mL. Straws were then left at room temperature (20-21°C) for 10min and subsequently submerged horizontally into the liquid nitrogen (Isachenko et al. 2012 J. Androl. 33, 462-468; https://doi.org/10.2164/jandrol.111.013789) and stored similarly to the conventionally cryopreserved straws. To thaw, vitrified straws were immersed in a water bath (42°C) for 20s. The DNA fragmentation was analysed using the APO DIRECT kit (BD PharmingenTM). The cells were stained according to the manufacturer's protocol, followed by flow cytometry analysis CyFlow (Sysmex-Partec). An analysis of variance with a significance of 0.05 for nonparametric statistical analysis to establish differences between groups was used. In our study, no statistically significant differences were observed in the total motility, progressive motility, or velocity parameters of spermatozoa (P&gt;0.05) post-thawing. Also, higher percentages of DNA fragmentation (35.1±8.1% vs. 20.1±6.8%; P&lt;0.05) were found in spermatozoa cryopreserved by means of vitrification with sucrose compared with conventional cryopreservation. Therefore, these methods are comparable and either can be implemented for the storage of spermatozoa to be used for future assisted-reproduction-technology procedures. Vitrification of human spermatozoa provides a simpler, faster, more cost-effective alternative to conventional cryopreservation methods.

Time-dependent effect of ground water fluoride on motility, abnormality and antioxidant status of spermatozoa: An in vitro study

The present study was undertaken to investigate the toxic effect of ground water fluoride (F) on motility, abnormality, and antioxidant status of spermatozoa. Treatment of ground water F with epididymal sperm suspension caused complete loss of sperm motility and decrease in the activities of superoxide dismutase (SOD) and catalase (CAT) and increase in the concentration of malondialdehyde (MDA) and abnormality of spermatozoa at 15 and 30 min time intervals. Further, incubation of spermatozoa with ground water F for 5, 10, and 15 min time intervals significantly reduced the sperm motility and activities of SOD and CAT and increased the concentration of MDA and abnormality of spermatozoa. The study revealed that F-induced effect on sperm motility and antioxidant status is time dependent. Increase in oxidative stress and concomitant decrease in motility of spermatozoa in ground water F clearly indicates that F-induced oxidative stress affected the sperm motility. The present study for the first time demonstrated the toxic effect of ground water F on spermatozoa at shorter duration of exposure, which affects the capability of spermatozoa in fertilization.

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&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.

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

Background: 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. Methods: 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: 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. Conclusions: 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.

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.

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<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.