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Images indicating bull spermatozoa viability determined using Nigrosin-eosin staining method. (A) live cell: head stained white; and (B) dead cell: head stained pink.
Source publication
The aim of this study was to evaluate the viability of bull spermatozoa diluted with commercial semen extender and two culture media stored at controlled room temperature (24 degrees C) for 72 hours. Two Nguni bulls were used for semen collection with the aid of an electro-ejaculator. After macroscopic evaluation, semen was pooled and aliquoted ran...
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The objective of our study was to determine the effect of 5% of CO 2 atmosphere and time of sample dilution on results of i n vitro analysis of stallion semen. Frozen-thawed semen from 14 stallions was incubated either in incubator or in a water bath, diluted prior to analysis or immediatelly after thawing. The following qualitative parameters were...
The aim of this study was to evaluate the effect of antioxidant agents and freezing methods on the ability of ram sperm to preserve its post-thaw quality characteristics. Six Chios rams were subjected to 52 weekly semen collections. Each ram was used as semen donor for freezing experiments once every 2 weeks. Equal number of good quality spermatozo...
Male germ cells have the potential to generate reactive oxygen species (ROS) during different stages of differentiation, laboratory manipulation and during their interaction with dissolved oxygen in extender. These ROS and its subset reactive nitrogen species (RNS) perform a concentration specific bimodal role, favouring oxidative eustress at physi...
Tmem95 encodes a sperm acrosomal membrane protein, whose knockout has a male-specific sterility phenotype in mice. Tmem95 knockout murine sperm can bind to, but do not fuse with, eggs. How TMEM95 plays a role in membrane fusion of sperm and eggs has remained elusive. Here, we utilize a sperm penetration assay as a model system to investigate the fu...
Citations
... An optimal diluent composition is expected to protect spermatozoa from damage caused by cooling, such as cryodamage or cryoinjury [6][7][8]. Liquid semen is considered successful if the decrease in motility (M) and metabolic activity due to cooling can be reversible [9]. ...
Background and aim:
Egg yolk (EY) is commonly used as an extracellular cryoprotectant in semen diluents but has some negative effects. Therefore, this study aimed to assess the potential of lecithin derived from plants, such as soybeans, as an alternative extracellular cryoprotectant and to characterize liquid semen quality of Ongole crossbred bulls using a modified caudal epididymis plasma-3 [CEP-3 (m)] as a base diluent and aqueous soybean extract (ASE).
Materials and methods:
A bull with progressive motility (PM) of fresh semen >70% was used. Two soybean extracts were also used, namely, ASE 1 and ASE 2, obtained by extraction procedures 1 and 2, respectively. The study was conducted using an experimental design with 11 treatments and ten replications, with diluents comprising different levels of ASE 1 and ASE 2, as well as a positive control with 10% EY. The parameters measured were motility (M) and its kinetic parameters, including PM, M, velocity curve linear, velocity straight linear, velocity average pathway, linearity, straightness, wobble, amplitude lateral head beat cross frequency, and hyperactivity using computer-assisted sperm analysis, viability, and spermatozoa abnormalities.
Results:
The CEP-3(m) diluent formula and ASE 1 at a 30% level maintained the PM of spermatozoa up to day 5 (40.7% ± 16.1%) of cold storage. Meanwhile, the CEP-3(m) diluent formula and ASE 2 could only maintain PM >40% until day 3 (42.1% ± 13.5%) of cold storage at a 30% level. The CEP-3(m) diluent and ASE 1 at a level of 25%-30% supported spermatozoa life (viability) up to day 5 with a value >80% (81.8 ± 3.5; 86.4 ± 2.6). The abnormality value of spermatozoa in various diluents during cold storage on days 0-5 was below 20%.
Conclusion:
Soybean extracts 1 and 2 can substitute EYs as extracellular cryoprotectants in modified CEP-3 basic diluents. Soybean extract 1 can support the life of spermatozoa up to day 5 but may cause the viscosity and movement of spermatozoa to be hyperactive. Soybean extract 2 can support the life of spermatozoa up to the 3rd day of cold storage and produces progressive (non-rotating) movement patterns. Further, research is recommended with higher levels of ASE 2.
... Semen storage at 25⁰C or 5⁰C for 30 minutes did not significantly affect spermatozoa viability but negatively affected spermatozoa motility (Alkali et al., 2022). Cell damage can cause spermatozoa death and be a source of toxicity for live spermatozoa (Raseona et al., 2017). ...
... Storage of spermatozoa at ambient temperature causes an increase in the rate of cell metabolism resulting in a deficit of energy sources, ATP, and accumulation of lactic acid and AAAO enzymes, so that the survival life of spermatozoa at ambient temperature (25-27°C) is short (Maulana, 2016;Raseona et al., 2017). The energy deficit causes an ATP deficit which affects the movement of the spermatozoa's tail so that the spermatozoa's motility decreases. ...
Artificial insemination with fresh semen is an alternative that can be done if there is a limitation of liquid nitrogen, bull, and facilities for processing liquid and frozen semen(costly). This study aimed to determine the optimal diluent formula based on CEP-3 (modified/m) and aqueous soybean extract (ASE) to support spermatozoa motility at ambient temperature storage. The research design was completely randomized, with eight treatments and six replications. The diluent formula treatments were as follows: T1 (CEP-3(m)), T2 (CEP-3(m)+10% Egg Yolk/EY), T3 (CEP-3(m)+ASE 1%), T4 (CEP-3 (m)+ASE 2%), T5 (CEP-3(m)+ASE 3%), T6 (CEP-3(m)+ASE 4%), T7 (CEP-3(m)+ASE 5%), and T8 (CEP-3(m)+ASE 6%). The parameters were measured, i.e., the motility of spermatozoa and itskinetic parameters.Data were analyzed using ANOVA with SPSS 16 program. The result showed that the progressive motility of spermatozoa at day one was more than 50% in diluents CEP-3(m)+ASE 1-2%. Diluent CEP-3(m) and aqueous soybean extract 1-2% can support spermatozoa's motility and kinetic parameters during storage at ambient temperature, including progressive motility, motility, motility, velocity, and BCF.
... To the best of our knowledge, very little information is available about the correlation between the extender type, BBE levels, and enzymatic activities in ram seminal plasma. Hence, the transaminase activities (AST and ALT) in extenders are a good indicator of semen quality because they measure sperm membrane stability [62]. For instance, ALT, AST, and ALP are fundamental for metabolic procedures that provide the energy necessary for sperm motility, viability, and fertility [63]. ...
Bee bread has numerous nutritional benefits and bioactive compounds. Other bee by-products have been used as extender additives to improve semen cryopreservation. Here, we examined the effects of supplementing egg yolk extender (EYE) or soybean lecithin extender (SBLE) with bee bread extract (BBE) on the quality of cryopreserved ram semen. Semen was collected from five adult Rahmani rams once a week for 7 weeks. EYE and SBLE were supplemented with BBE. Antioxidant capacity and total phenolic compound, total flavonoid compound, and total soluble carbohydrate levels of BBE were measured. Sperm characteristics, including progressive motility, viability, abnormalities, membrane integrity, and acrosome integrity, were analyzed after equili-bration, thawing, and thawing followed by a 2-h incubation. The total antioxidant capacity and malondialdehyde, hydrogen peroxide, aspartate transaminase, alanine transaminase, alkaline phosphatase, and total acid phosphatase levels in extenders were determined after thawing. Sperm apoptosis was analyzed using annexin V assays. SBLE was more effective than EYE for cryo-preserving ram semen. Extender supplementation with BBE improved ram semen quality during freezing in a concentration-dependent pattern. Motility, vitality, and membrane integrity were particularly enhanced in BBE-treated semen. Additionally, BBE promoted antioxidant and enzy-matic activities and reduced apoptosis in semen. Thus, extender supplementation with BBE improved sperm cryopreservation.
... Eosin-nigrosin stain was used for assessment of sperm viability as described by Raseona et al. [15]. After staining, the slides were dried and covered with a cover slip before evaluation by using CASA at 60× magnification. ...
Postthaw survival of spermatozoa with high motility and vitality is required for successful artificial insemination. The aim of this study was to compare the effects of two different thawing methods on motility, morphology, kinematic parameters, and viability of spermatozoa with a computer-assisted semen analyzer (CASA). Frozen bull spermatozoa were thawed by using two different thawing procedures: 1) Water bath; straws were thawed in a water bath at 37 °C for 30 s. 2) Dry thawing system; straws were thawed in a dry thawing device at 37 °C for 30 s. A total of 10 straws were used for each thawing procedure. There were significant differences between the thawing methods for acrosome defects (P < 0.05), head defects (P < 0.0001), middle part defects (P < 0.05), and total abnormal spermatozoa rates (P < 0.05). There was no difference between the thawing methods for total and progressive sperm motility determined with CASA. There were significant differences (P < 0.05) between the thawing methods for STR and ALH values which were kinematic parameters. The STR (66.37%) and ALH (3,28 μm) values of dry system were higher (P < 0.05) than the STR (57.88%) and ALH (2.78 μm) values of water bath. In conclusion, the present study demonstrated the possibility of using dry thawing system as alternative to water bath for thawing bull sperm because some postthaw sperm values obtained when dry thawing system was used were better than those obtained when water bath was used.
... Our results agree with these observations because the use of Biladyl (egg-based medium) showed lower velocity and linearity than when Andromed (lecithin-based medium) was used. In an apparent contradiction, it has been reported that VCL is higher with the use of egg yolk (Triladyl) than with egg yolk-free media (TCM-199 and Ham's F-10), but these results were obtained using a different bull breed (Raseona et al., 2017). ...
The evaluation of sperm motion is crucial for processing of seminal doses for artificial insemination. Here, the combined effect of the type and capture area of three counting chambers, together with the type of diluent employed, on sperm motility was analysed. Ejaculates from thirteen Holstein bulls were used for sperm kinematic analysis with the ISAS®v1 CASA‐Mot system, using two capillary‐loaded counting chambers (Leja® and Cell‐Vu®) and one drop displacement chamber (Makler®). Nine fixed positions were analysed per chamber type, considering central and lateral and three longitudinal fields. Independent of the diluent used, differences were found between the three chambers. Independent of the extender, no differences in x‐axis were observed with Cell‐Vu®, while using Leja®, some parameters showed lower values in the centre than in lateral areas. In both counting chambers, the lowest values were observed in the distal area. Results obtained with the two diluents were highly different with a very low correlation between them. In conclusion, the capture area inside the chambers leads to significant changes in sperm kinematic parameters and different dilution media introduce considerable differences in the motility patterns. It is necessary to optimise sampling methods and specific set‐ups to be used with CASA‐Mot technology.
The present study was undertaken to assess viability of frozen-thawed bull semen collected from the bull's ejaculate and cauda epididymis. A total of 30 ejaculates were collected from three bulls twice per week for 5 weeks (Control). Caudal epididymis were collected from slaughtered beef cattle of unknown origin from the local abattoir. Caudal epididymal sperm was recovered immediately after slaughtering (EP-0 h) and after cooling at 5°C for 24 h (EP-24). The epididymal and ejaculated samples were each pooled together before being extended with Triladyl. Diluted samples per treatment were loaded into a 0.25-mL French straw and cooled to 5°C in 4 h. Cooled straws were placed 4 cm above liquid nitrogen to freeze for 10 min. Frozen straws were immersed into LN2 and kept for 7 days at -196°C. Samples were analysed immediately after dilution and post-thawing using the computer aided sperm analysis for sperm motility rate, viability and acrosome defects. The highest sperm motility rates were observed with EJ-0 h before and after cryopreservation. However, the difference in sperm motility parameters between EP-0 h and EP-24 h evaluated before and after freezing was not significant (P > 0.05). Furthermore, no significant difference in live cells mean values was observed between the three samples on freezing (P > 0.05). In relation to spermatozoa acrosome defects, there was no significant difference observed among the three samples before and on freezing (P > 0.05). In conclusion, the results from the present study revealed that cooling of epididymides at 5°C for 24 h before the recovery of sperm cells was efficient in preserving epididymal sperm viability. However, ejaculated bull spermatozoa had higher sperm motility and viability rate than epididymal sperm.
