Post Thaw Research Articles

Supplementation of Coenzyme Ubiquinone (COQ10) in the semen diluent of Balitbangtan’s Superior Native Chicken in Terms of Spermatozoa Cromatin Damage

The development of technology in the field of animal husbandry is growing very rapidly, apart from ruminants from poultry livestock can also be done artificial insemination used for breeding. According to Apriyanti (2017) The success of the mating system can be seen from the success of livestock mating which is influenced by the quality of the semen produced. One of the supplementations used is coenzyme ubiquinone (CoQ10) where CoQ10 is one of the vitamins such as fat-soluble vitamin E which is endogenously present in the inner membrane of mitochondria of mammals and plants (El-Sayed, et al. 2021). This study used an experimental method or field experiment with 4 treatments and 10 replicates on 4 male KUB chickens aged 12 months and the diluent used was egg yolk lactated ringer. The variables observed in this study wes cromatin damage of spermatozoa before freezing and post thawing. The results obtained in this study chromatin damage before freezing obtained the highest average value of 1.18 ± 0.58 with statistical calculations showed significantly different results (P < 0.05). chromatin damage after thawing back obtained the highest result of 2.80 ± 0.89 with statistical test calculations obtained results that were not significantly different (P < 0.01) to the different levels of Coenzyme ubiquinone in diluents with different levels.

Cryoprotective Effect of Zinc and Gold Nanoparticles During Cooling and Freeze-thawing on Marwari Stallion Sperm Parameters and Reactive Oxygen Species Production

BACKGROUND: Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Different substances and compounds can be added to semen extenders to improve sperm quality. OBJECTIVE: To investigate the effect of supplementing semen extender with zinc nanoparticles (ZnONPs) and gold nanoparticles (AuNPs) on cooled and frozen-thawed spermatozoa of Marwari stallion. MATERIALS AND METHODS: A total of 20 ejaculates from four Marwari stallions (five ejaculates from each) were collected. The gel free semen was extended with primary extender in equal volume and then divided in three equal groups, namely control (C), zinc (ZnO) and gold (Au), and centrifuged to obtain a final concentration of 100-150 x10 6 mL -1 and then cryopreserved. Cooled and post-thaw semen evaluations were conducted for various seminal characteristics, viz. progressive sperm motility, sperm plasma membrane integrity, sperm viability, acrosome integrity and mitochondrial membrane potential. RESULTS: Cooled semen (4ºC for 2 h) evaluation revealed nonsignificant differences among the groups (C, ZnO and Au) for all the semen quality parameters. However, at the post-thaw stage, progressive sperm motility, sperm plasma membrane integrity, acrosome integrity and oxidative parameters were significantly (P≤ 0.01) higher in the ZnO group than Au and C. CONCLUSION: The addition of ZnO nanoparticles improves the post thaw quality of stallion spermatozoa by reducing the oxidative stress, but Au nanoparticles had no effect on cooled as well as post-thaw semen quality.

Impact of controlled ice nucleation on intracellular dehydration, ice formation and their implications on T cell freeze–thaw viability

Cryopreservation is important in manufacturing of cell therapy products, influencing their safety and effectiveness. During freezing and thawing, intracellular events such as dehydration and ice formation can impact cell viability. In this study, the impact of controlling the ice nucleation temperature on intracellular events and viability were investigated. A model T cell line, Jurkat cells, were evaluated in commercially relevant cryoformulations (2.5 and 5 % v/v DMSO in Plasma-Lyte A) using a cryomicroscopic setup to monitor the dynamic changes cells go through during freeze–thaw as well as a controlled rate freezer to study bulk freeze–thaw. The equilibrium freezing temperatures of the studied formulations and a DMSO/Plasma-Lyte A liquidus curve were determined using DSC. The cryomicroscopic studies revealed that an ice nucleation temperature of −6°C, close to the equilibrium freezing temperatures of cryoformulations, led to more intracellular dehydration and less intracellular ice formation during freezing compared to either a lower ice nucleation temperature (−10 °C) or uncontrolled ice nucleation. The cell membrane integrity and post thaw viability in bulk cryopreservation consistently demonstrated the advantage of the higher ice nucleation temperature, and the correlation between the cellular events and cell viability.

Use of a sperm-Hyaluronan binding assay for evaluation of sperm quality in dromedary camels

The objective of this study was to assess the ability of camel spermatozoa to bind in the Hyaluronan Binding Assay (HBA), to determine if conventional sperm quality parameters, in vitro fertilization capacity, and precursor of A-Kinase Anchoring Protein 4 (proAKAP4) values correlate with HBA results. The potential to predict post-thaw fertilization performance from HBA for fresh dromedary camel sperm was also evaluated. Semen samples were collected and assessed both fresh and post thawing, at 0 h and 1.5 h. Conventional semen analysis, HBA, and a proAKAP4 biomarker-test were used to validate sperm quality. A heterologous sperm penetration assay using zona pellucida-free goat oocytes was used to assess in vitro sperm fertilizing capacity. The results showed that dromedary camel spermatozoa bound to hyaluronan with no correlation between results from fresh samples and after thawing. Furthermore, the proAKAP4 test results showed a negative correlation with HBA at 0 h after thawing (r = - 0.62; P = 0.03). In the conventional analysis, only progressive motility (r = 0.65; P = 0.02) and straightness correlated with HBA for fresh semen (r = 0.69; P = 0.01). In the sperm penetration assay, a moderate but non-significant correlation was identified between fresh sperm HBA and penetration (r = 0.52; P = 0.07). In conclusion, results suggested that HBA can be used to assess camel sperm properties, but further investigation is needed to understand its correlation with other sperm quality parameters. The HBA score from fresh dromedary camel sperm was unable to predict post-thaw fertilization performance.

The characteristics of frozen-thawed rooster sperm using various intracellular cryoprotectants

Cryopreservation of rooster semen is essential for conserving genetic resources, genetic improvement, and increasing productivity. However, the nature of avian sperm presents a global issue in ensuring superior frozen semen for artificial insemination. Thus, the present study aimed to evaluate the impact of using dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and ethylene glycol (EG) as cryoprotectants on post-thawed sperm motility, quality, antioxidant indicators, and fertilizing capacity. Twice a week, fresh semen ejaculates were collected from 15 adult roosters and immediately evaluated to constitute a pool from clean and qualified samples. The pooled semen was further diluted at a ratio of 1:2 (v/v) with an extender and then subjected to a freezing protocol in a liquid nitrogen vapor after adding a cryoprotectant solution containing 6% of either DMA, DMSO, or EG, respectively. After thawing, characteristics of sperm motion, quality, antioxidants, and fertilizing ability were evaluated and compared to fresh and cooled semen as controls. The results demonstrated that semen cooling negatively affected some parameters of sperm motility, quality, antioxidant biomarkers, and fertility. In comparison to the DMSO and EG groups, employing DMA considerably (P < 0.05) raised the percentages of sperm progressive motility, viability, plasma membrane intactness, and DNA integrity. The DMA group showed a significant increase in the catalase and glutathione reduced antioxidant enzyme activity and a reduction in nitric oxide and lipid peroxidation. After artificial insemination, the DMA and DMSO groups exhibited considerably (P < 0.05) better rates of hatchability and fertility than the EG group. It is concluded that freezing extenders containing 6% DMA is better than DMSO or EG to improve the post thaw semen quality and fertility in chickens.

P-239 Comparative study between two commercial vitrification kits on survival and clinical outcome for blastocyst frozen embryo transfer

Abstract Study question To compare the post thaw survival and clinical outcome of blastocyst (D5/D6) transfer between two different commercially available kits for freezing and thawing. Summary answer There is significant difference on survival of post thaw blastocyst and clinical outcomes between Kitazato (Japan) and Irvine (USA) vitrification media. What is known already Vitrification is widely used in embryo preservation for its reliable outcome and the ease of use. Different brands of commercial freezing and thawing kits are available. The main difference between the two commercial vitrification kits used in this study are the different extracellur cryoprotectant used in which Kitazato contain trehalose supplemented with hydroxypropyl cellulose while Irvine contain sucrose with dextran serum supplement. Recent study suggested that cell apoptosis in specimen vitrified in trehalose media is lower in comparison to sucrose. Study design, size, duration Retrospective study was carried out from 2022-2023. Patient with uterine abnormalities, history of recurrent implantation failure (RIF), patient that underwent Preimplantation Genetic Testing (PGT) and those transferring D2/3 embryo were excluded. Survivability of post thaw blastocyst and clinical outcome of frozen blastocyst transfer were compared between Kitazato (N = 374) and Irvine (N = 253) vitrification kit. The data was analyzed using t-test and Anova. Participants/materials, setting, methods Blastocysts that were vitrified and warmed with vitrification kit from the same manufacturer were grouped together and compared. Patient in each vitrification arms were divided into two main age groups which are &amp;lt;37 years old and ≥37 years old. The survivability and clinical outcome of the two respective vitrification kits was compared between the age group, blastocyst grading prior to freezing (Good, Fair, Poor) and the day of blastocyst freezing (D5 or D6). Main results and the role of chance The overall mean post thawed survival was higher in Kitazato (95.8%) compare to Irvine (91.8%) (p &amp;lt; 0.001). In patient age &amp;lt;37 years old, Kitazato group performed better when compare to Irvine group. Post thawed survival using Kitazato warming was significantly better (p &amp;lt; 0.001) on good (AA,AB,BA) and fair graded (BB) blastocyst with mean survival of 97.1% vs 93.4% and 94.9% vs 87.6% respectively. Similarly, post thawed survival of D5 and D6 blastocysts were significantly higher in Kitazato group when compared to Irvine group with mean survival of 96.7% vs 92.8% and 94.8% vs 90.3% respectively. However no significant difference was observed in post thawed survival outcome for patient aged ≥ 37 years old between both groups in the same study parameters. In terms of clinical outcomes, there is also no difference observed between Kitazato group and Irvine group on beta hCG positive rates. Interestingly, the Irvine group shown to have significantly higher clinical pregnancy compare to Kitazato group (53% vs 60% respectively) but no significant difference was observed in sub group for patient aged &amp;lt; 37 years old and ≥ 37 years old. Furthermore, the implantation rate (IR) in Irvine group group (55.7%) is also significantly higher compare to the Kitazato group (50.5%). Limitations, reasons for caution Potential confounder factors such as stimulation protocol and the embryologist experience were not factored in may have affected the outcome of the study. Sample size can be increased to improve confidence in this study. Wider implications of the findings This study demonstrates that the post thaw survivability is better in the Kitazato group while the clinical outcome is better in the Irvine group. Thus this can give a better understanding of the respective commercial vitrification kits and provide insight for laboratories to choose for their clinical practices. Trial registration number Not Applicable

P-347 In search of the most efficient device to freeze low numbers of spermatozoa: a comparative evaluation of various vitrification device

Abstract Study question Can different vitrification devices be used for cryopreservation of few spermatozoa for patients with varying sperm concentrations and which device is the most efficient? Summary answer The study demonstrates the efficacy of Cryotop, CBS, and Vitrifit devices in cryopreservation of low numbers of spermatozoa, with Cryotop Open showing superior performance. What is known already Sperm cryopreservation is crucial for managing male-factor infertility, yet conventional methods, reporting significant reduction in motility with only 30 to 40% viability post-thawing, fall short of optimal cryopreservation of small numbers of spermatozoa. Currently, ongoing efforts to enhance cryopreservation techniques seek to minimize cryodamage and optimize outcomes, particularly for spermatozoa from patients with conditions like oligozoospermia, cryptozoospermia, or azoospermia, where each spermatozoa is of substantial importance. Several devices have shown promising results regarding cryopreservation of small numbers of spermatozoa. However, to date, no comparison has been performed between multiple embryo-vitrification devices in terms of recovery and viability post thawing. Study design, size, duration Between January 2023 and May 2023, this study included 50 fresh ejaculated routine samples: 25 with spermatozoa concentrations &amp;gt;1 million/ml and 25 samples with concentrations &amp;lt;1 million/ml. For each sample, 10 motile spermatozoa were loaded on four different embryo-vitrification devices performed in triplicate: Cryotop Open (Kitazato), Cryotop Closed (Kitazato), CBS (Cryo BioSystem) and Vitrifit (Cooper Surgical). Recovery, motility, immotile vitality rates, user-friendliness as well as the duration of the freezing/thawing procedures were examined. Participants/materials, setting, methods Ten spermatozoa, selected in Hepes-HSA, were loaded into a 2 µl mixture of SpermFreeze and HEPES-HSA. Held 3 cm above LN2 for 2 minutes, straws were plunged into LN2. Thawing involved immersing straws in 2 µl Hepes-HSA under oil. Hypo-Osmotic-Swelling test was used for the vitality assessment of the immotile spermatozoa. Thawing protocols showed minor variations based on the carrier type. The study was approved by the local Ethics Committee. Main results and the role of chance This study conducted a detailed analysis of four vitrification devices for cryopreservation of low spermatozoa numbers. For concentrations &amp;gt;1 million/ml, Vitrifit, Cryotop Open, Cryotop Closed, and CBS displayed comparable recovery rates (mean % ± SD) (85±0.09, 89±0.06, 84±0.10, 86±0.07); motility rates (76±0.12, 79±0.08, 78±0.13, 80±0.08); immotile vitality rates (16±0.08, 13±0.07, 13±0.09, 12±0.07). In this group, Vitrifit and Cryotop Open exhibited similar freezing and recovery times (4’10’’/7’42’’ and 4’11’’/7’40’’ respectively). Cryotop Closed and CBS had slightly longer freezing times (4’33’’ and 5’31’’ respectively), with Cryotop Closed showing the fastest recovery (7’11’’). Similarly, for sperm concentrations &amp;lt;1 million/ml, devices demonstrated similar recovery (87±0.09, 91±0.06, 88±0.07, 83±0.12), motility (55±0.10, 61±0.09, 56±0.07, 50±0.09), and immotile vitality (26±0.07, 26±0.06, 27±0.07, 27±0.06). For concentrations &amp;lt;1 million/ml, all devices showed similar freezing and recovery times. Statistical analyses, including ANOVA and Levene’s test, revealed no significant inter-device differences, substantiated by minimal Cohen’s d effect sizes. Regarding the practical aspect, due to the ergonomic design and plastic composition that allows better microscopic visibility, Cryotop (both closed and open) demonstrated superior handling performance to CBS. Considering both statistical validation and manipulation, Cryotop Open distinguishes itself as the most efficient and user-friendly device for cryopreservation of low spermatozoa numbers. Limitations, reasons for caution The study’s main limitation is the limited sample size. Further research with larger cohorts and exploration of fertilization potential is necessary to validate these findings. Additionally, our study did not explore potential variations in cryopreservation outcomes related to additional sperm sample characteristics, which could impact the results. Wider implications of the findings Successful cryopreservation of low sperm numbers provides hope for cryptozoospermia and (non-)obstructive azoospermia patients. It allows the use of their own sperm, potentially avoiding donation. With superior frozen sperm survival, recovery rates, and a shorter search time, this method enhances fertility preservation accessibility, guiding patients in future IVF treatments effectively. Trial registration number EC-2022-272

Seminal characteristics and efficacy of egg yolk free semen extender on cryopreservation of Murrah buffalo bull semen

The experiment was undertaken to study the efficacy of egg yolk free semen extender on cryopreservation of buffalo semen. A total of 24 semen ejaculates from Murrah bulls (5 to 6 years) maintained at Central Semen Station, Anjora, Durg, Chhattisgarh were divided into two aliquots and were diluted using Tris-fructose-egg yolk-glycerol (TFYG) and egg yolk free commercial extender (EYFCE, AndroMed). The overall average Murrah bull fresh semen volume (mL), sperm concentration (million/mL), initial progressive motility (%), live sperm (%), abnormal spermatozoa (%), intact acrosome (%) and HOS reactive sperm (%) were 4.90±0.49, 1286.92±100.23, 75.00±0.67, 81.45±0.61, 10.70±0.51, 80.16±0.43 and 68.83±0.40, respectively. There was significant difference between bulls in semen volume (P&lt;0.01), individual motility (P&lt;0.01) and total sperm abnormalities (P&lt;0.05). The semen extended with TFYG and EYFC extenders were cryopreserved in french mini-straw (0.25 mL) using programmable bio-freezer. The mean values of post thaw motility (%), live sperm (%), abnormal sperm (%), intact acrosome (%), HOST (%) of Murrah bull semen extended with TFYG and EYFCE were 45.83±0.77 vs 49.16±1.33, 57.41±0.54 vs 58.62±0.73, 12.54±0.46 vs 12.45±0.63, 69.62±0.82 vs 71.45±0.64 and 56.45±0.52 vs 57.37±0.55, respectively. Post thaw motility, live spermatozoa and intact acrosome were significantly higher (P&lt;0.01) in frozen thawed semen extended with as EYFCE compared to TFYG in Murrah bull semen. It is concluded that EYFCE results in improvement in post thaw semen characteristics viz. post thaw motility, live sperm percent and intact acrosome.

Differences in analytical methods affect the analytical values of soluble solids, free sugar, and organic acid content of strawberries

The present study compares different pre-treatments used to analyze free sugars and organic acids in strawberries. Soluble solid content, free sugars, and organic acid content in strawberries may vary depending on the pre-treatment methods used for analyze. A comparison of freshly squeezed juice and that obtained post freezing and thawing revealed a significant reduction in sucrose content in the once-frozen juice. However, no significant differences were observed in the soluble solids, glucose, and fructose contents. Furthermore, crushed flesh had significantly higher soluble solid, glucose, and fructose levels and lower sucrose content than those of the juice. Crushed flesh exhibited significantly higher citric acid and a lower malic acid content than those in the juice. There was a significant reduction in sucrose content in the freeze-dried samples compared to all other pre-treatments. Contrastingly, citric and malic acid levels were higher in the freeze-dried samples than that in the freshly crushed samples. The sucrose analytical values can be greatly decrease during the pre-treatment process. Therefore, differences in analytical methods should be considered when comparing sucrose content and composition.

Temporal dynamics of in vitro hemostatic function in platelets cryopreserved using a novel approach for rapid issuance.

Current procedures for thawing and issuing of cryopreserved platelets (CPPs) are laborious and have remained challenging in emergency settings such as blood banks and military operations. In this prospective study, a novel processing method designed to facilitate the rapid issuance of CPPs with no postthaw handling required was developed and functionally characterized in parallel with standard CPPs manufactured. Double-dose plateletpheresis units (n = 42) were cryopreserved at -80°C in 5%-6% dimethyl sulfoxide to produce matched pairs thawed successively over a 27-month period for comparison between two processing arms. In contrast to the standard CPPs manufactured as standalone units, platelets were frozen in tandem with resuspending plasma in a distinct partition as a single unit in the novel method, herein referred to as tandem CPPs. Postthaw (PT) CPPs from both arms were assessed at PT0-, 12-, and 24-h to measure platelet recovery, R-time (time to clot initiation; min), and maximum amplitude (MA; clot strength; mm) using thromboelastography. In the overall dataset, mean platelet recovery was higher (p < .0005) for tandem CPPs (83.9%) compared with standard CPPs (73.3%) at PT0; mean R-times were faster (p < .0005) for tandem CPPs (2.5-3.6 min) compared with standard CPPs (3.0-3.8 min); mean MA was higher for tandem CPPs (57.8-59.5 mm) compared with standard CPPs (52.1-55.8 mm) at each postthaw time point (p < .05). Robust temporal dynamics of superior hemostatic functionality were established for tandem CPPs over extended cryopreservation up to 27 months and 24 h of postthaw storage.

Influence of Method Thawing to Success Artificial Insemination of Cows

Artificial insemination has been widely known among breeders as an effective reproductive technology by inserting sperm collected from male animals into the reproductive tract of female animals for the purpose of improving genetic quality. The success of AI is influenced by several factors, namely semen quality, inseminator expertise, livestock health, and other supporting factors. Good semen quality is one of the determining factors for the success of artificial insemination. One of the quality standards for frozen semen according to SNI is the level of motility post thawing minimum 40%. Motility level post thawing very dependent on technique thawing used. Thawing is the process of re-thawing frozen semen at physiological body temperature to reactivate sperm cells. Thawing can have an impact on reducing the quality of spermatozoa if not done properly. Temperature and time accuracy thawing will maintain sperm viability and motility, so that sperm quality is maintained and the success rate of artificial insemination will be high.

Post-thaw quality of boar spermatozoa is affected by ejaculate fractions and extenders.

The aim of this study was to investigate the effect of different extenders on the post-thaw (PT) quality of sperm originating from the sperm-rich fraction (SRF) and post-sperm-rich fraction (PSRF) of boar ejaculate. Motility and velocity parameters, analyzed using a computer-assisted semen analysis (CASA) system, and membrane integrity parameters were markedly higher in frozen-thawed (FT) spermatozoa of the SRF in both the Belstville Thawing Solution (BTS) and Androhep Plus (AHP) extenders, irrespective of the post-thaw (PT) storage time. Furthermore, reduced cryo-survival was more marked in FT spermatozoa of the PSRF in both extenders following storage for 60 min. It was found that the SRF-stored samples in the AHP extender for 60 min exhibited significantly higher percentages of spermatozoa with total motility, mitochondrial function and acrosome integrity than those stored in the BTS extender. The findings of this study confirm that components of the ejaculate fractions and extender have varying effects on the cryo-survival of boar spermatozoa.

A droplet vitrification cryopreservation protocol for conservation of hops (Humulus lupulus) genetic resources

Hops (Humulus lupulus L.) is essentially used in the brewing industry as it contributes to flavor, and aroma of beer. However, the genetic diversity of hops is increasingly threatened by diseases, environmental changes, and urbanization. Cryopreservation has emerged as a pivotal strategy for safeguarding and maintaining the genetic diversity of hops. The present work presents a comprehensive study on the cryopreservation of hops, focusing on the development and optimization of a droplet vitrification based cryopreservation protocol. Shoot tips excised from one month old in vitro cultures were precultured on 0.3 M sucrose, dehydrated in a loading solution followed by treatment with PVS2 solution for different durations. Significant effect of PVS2 dehydration was observed on post-thaw survival and regeneration after cryoconservation with maximum 50% post-thaw regeneration observed in shoot tips dehydrated in PVS2 for 30 min. Genetic fidelity of the regenerated plants was confirmed using 30 ISSR markers. Reproducibility of the developed protocol was tested on seven other accessions and post thaw regeneration ranging from 43 to 70% was observed across the accessions. The present study reports a highly efficient protocol for conservation of hops germplasm. The results indicate that droplet vitrification can be used as a reliable and sustainable approach for hop genetic preservation, with high survival rates and minimal genetic alterations observed in cryopreserved samples. To the best of our knowledge, this is the first report on DV based cryopreservation of hops germplasm.

Post-thaw sperm morphokinetics and mitochondrial genes profile of buck semen supplemented with selenium in two different forms

Background The quality characteristics of semen is a determinant factor for improving fertility of rabbit. Objectives The current investigation was done to evaluate post-thaw sperm morphokinetics and mitochondrial genes expression profile of buck semen supplemented with selenium in two different forms. Materials and methods Sexually mature bucks (n=16) that aged three months on average were used for collecting two ejaculates which were evaluated using computer assisted semen analysis (CASA) program. The semen samples of all experimental animals which recorded progressive motility greater than or equal to 70% were pooled for cryopreservation. The pooled semen was divided into three groups that were supplemented with selenium nanoparticles and selenium in normal form in addition to the control group. Morphological characteristics as well as CASA parameters were assessed after freezing for 1 week. Moreover, enzymatic activity assays were performed to measure the antioxidant capacity of cryopreserved buck semen. Transcriptional profile of mitochondrial activity and antioxidant defense regulating genes was conducted using quantitative real-time polymerase chain reaction (PCR). Results and conclusion The addition of selenium in normal and Nano forms has significantly (P ≤0.05) enhanced some of CASA parameters such as DCL (µm), DAP (µm), DSL (µm), VCL (µm/s), VAP (µm/s), Amplitude of lateral head (ALH) (µ), BCF (Hz), and VSL (µm/s) during prefreezing period compared with control group. Moreover, the data presented in the present study indicated a significant (P ≤0.05) improvement of post-thaw total and progressive sperm motility in the two groups supplemented with normal and Nano compared with control group. The post-thaw level of total antioxidant capacity (TAC) and percentage of live sperm were higher in the two groups supplemented with selenium and Nano selenium than the control group. The expression profile of candidate genes regulating mitochondrial activity (ATP Synthase F1 Subunit Alpha (ATP5A1), NADH dehydrogenase subunit1 (ND1), NADH dehydrogenase subunit2 (ND2), and Carnitine palmitoyltransferase (CPT2)) was increased significantly (P ≤0.05) in semen supplemented with selenium in normal and Nano forms compared with the control group. In conclusion, the data of this investigation demonstrated enhancement of CASA parameters during pre-freezing post thaw total and progressive sperm motility in the two groups supplemented with normal and nano selenium. The proportion of sperm viability and the level of total antioxidant capacity were enhanced in the two groups supplemented with selenium which was coupled with up-regulation of mitochondrial transcripts. It seems the both two forms shared the same mechanism on improving post-thaw physical and molecular qualities of rabbit sperm.

Evaluation of Metformin Supplemented Diluent on Cryopreservation of Canine Semen

Background: Cryopreservation of semen in canines is getting more advanced lately, however the fertility following the insemination of the frozen thawed semen is lower than other species. The higher content of PUFA and lipid peroxidation leading to production of reactive oxygen species is one of the reasons for the same. Antioxidants play a role in improving the post thaw semen quality in canines. Methods: Semen collected from 10 male dogs were further divided into two aliquots. The first aliquot was extended with TRIS-fructose egg yolk extender and the second one was extended with TRIS-fructose egg yolk extender supplemented with 50 µM of metformin. The extended semen was cryopreserved using conventional methods. Subsequently, both routine semen parameters and advanced semen parameters including viability, acrosomal integrity, DNA integrity and mitochondrial reactive oxygen species (ROS) were measured in the spermatozoa through the utilization of flow cytometry. Result: Present study indicated that, post thaw motility was significantly higher in the metformin supplemented group in comparison with control while, the mitochondrial ROS was significantly lower in metformin supplemented group. Our study concludes metformin could be effectively used to reduce the ROS produced during the process of cryopreservation of the canine semen.

Addition of low concentration of cholesterol-loaded cyclodextrin (CLC) has a positive effect on cryopreserved canine spermatozoa evaluated by andrological and biophysical methods

BackgroundThis study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP).ResultsAn addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P < 0.05). 1.66 mg/ml HBCD decreased progressive motility of spermatozoa and population with rapid movement relative to the control (P < 0.05). Furthermore, the groups with an addition of 1.66 mg/ml and 3.32 mg/ml of CLC, as well as the group with only cyclodextrin, increased percentage of dead spermatozoa without lipid peroxidation and decreased percentage of viable spermatozoa without LPO which was lower in these groups than in the control (P < 0.05). Other sperm parameters assessed on flow cytometer were not significantly different. The addition of CLC at 0.83 mg/ml and 3.32 mg/ml concentrations and 1.66 mg/ml of HBCD caused an increase in ANISO measured at 23 ºC (P < 0.05).ConclusionsIn conclusion, the results suggest that increasing cholesterol in the plasma membrane of canine spermatozoa can improve their freezability. However, only low concentrations of CLC may improve semen quality after thawing without adversely affecting other parameters.

Key quality parameter comparison of mesenchymal stem cell product cryopreserved in different cryopreservation solutions for clinical applications.

Cryopreservation is a critical process of cell products for achieving a commercial viability through wide scale adoption. By preserving cells in a lower temperature, cryopreservation enables a product to be off-the-shelf and ready for infusion. An optimized cryopreservation strategy can maintain the viability, phenotype, and potency of thawed mesenchymal stromal/stem cells (MSCs) while being regulatory compliant. We compared three clinical-ready formulations with one research cryopreservation solutions and evaluated key quality parameters of post thawed MSCs. MSCs were cryopreserved at 3, 6, and 9 million cells/mL (M/mL) in four different cryopreservation solutions: NutriFreez (10% dimethyl sulfoxide [DMSO]), Plasmalyte A (PLA)/5% human albumin (HA)/10% DMSO (PHD10), CryoStor CS5 (5% DMSO), and CryoStor CS10 (10% DMSO). To establish post thaw viability, cells were evaluated with no dilution of DMSO (from 3M/mL), 1:1 dilution (from 6M/mL), or 1:2 dilution (from 9M/mL) with PLA/5% HA, to achieve uniform concentration at 3M/mL. Cell viability was measured at 0-, 2-, 4-, and 6-h post thaw with Trypan blue exclusion and Annexin V/PI staining. Dilution (1:2) of final cell products from 9M/mL resulted in an improvement of cell viability over 6h but showed a trend of decreased recovery. MSCs cryopreserved in solutions with 10% DMSO displayed comparable viabilities and recoveries up to 6h after thawing, whereas a decreasing trend was noted in cell viability and recovery with CS5. Cells from all groups exhibited surface marker characteristics of MSCs. We further evaluated cell proliferation after 6-day recovery in culture. While cells cryopreserved in NutriFreez and PHD10 presented similar cell growth post thaw, MSCs cryopreserved in CS5 and CS10 at 3M/mL and 6M/mL showed 10-fold less proliferative capacity. No significant differences were observed between MSCs cryopreserved in NutriFreez and PHD10 in their potency to inhibit T cell proliferation and improve monocytic phagocytosis. MSCs can be cryopreserved up to 9M/mL without losing notable viability and recovery, while exhibiting comparable post thaw potency with NutriFreez and PHD10. These results highlight the importance of key parameter testing for selecting the optimal cryopreservation solution for MSC-based therapy.

Studies on Fresh and Cryopreserved Seminal Attributes in Beetal Bucks Outside their Breeding Tract

The objectives of the present research were to assess fresh and cryopreserved. seminal characteristics of breeding Beetal buck semen outside of its breeding tract. Semen was collected twice in a week from twelve Beetal bucks for 6 weeks. The mean value of ejaculate volume(ml), mass motility(o-5 scale), progressive motility(%), sperm viability (%), intact acrosome(%), sperm abnormality(%) and plasma membrane integrity(%) were ranged from 0.73 ± 0.08 ml to 1.17 ± 0.27 ml; 4.17+ to 4.67+, 80.83 ± 1.54 to 87.50 ± 2.14; 83.83 ± 0.95 to 90.33 ± 0.80; 87.00 ± 0.86 to 94.67 ± 0.33; 6.83 ± 0.31 to 10.33 ± 0.67 and 59.33 ± 1.52 to 66.67 ± 1.17, respectively. The mean value of post thaw sperm motility, post thaw sperm viability, post thaw acrosome integrity, post thaw sperm abnormalities and post thaw plasma membrane integrity were ranged from 52.50± 1.12 to 66.67 ± 1.67; 60.00 ± 1.24 to 69.67 ± 1.05; 66.00 ± 0.77 to 78.83 ± 2.15; 9.00 ± 0.45 to 12.83 ± 0.60 and 41.50 ± 1.18 to 53.50 ± 1.23, respectively. It was determined that, even outside of the breeding tract, the foundational characteristics of Beetal bucks are within the range defined for other Indian breeds of goat.

Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes

Adding endogenous antioxidants to the diluent is significantly associated with semen quality during the freezing process. This study aimed to investigate the effects of adding glutathione to AndroMed diluent on the preservation of crucial sperm attributes, namely, intact plasma membrane (IPM), intact acrosome membrane (IAM), and progressive motility of Bali cattle spermatozoa. A completely randomized design was used, and spermatozoa samples were obtained from a Bali cattle and divided into two diluent treatment groups (36 diluent samples in each group with six replications), namely pure AndroMed as the control and a group with the addition of glutathione (1 mmol) to AndroMed. Each treatment was replicated six times and evaluated at three freezing stages, including post-dilution, post-equilibration, and post-thawing, for crucial sperm properties. The results indicated that fresh Bali cattle spermatozoa had progressive motility, IAM, and IPM of 75%, 89%, and 88%, respectively. During the freezing process, there was a significant decrease in semen quality, including progressive motility, IAM, and IPM of spermatozoa after dilution to post-equilibration and post-equilibration to post-thawing in both treatment groups. Meanwhile, the addition of 1 mmol of glutathione to AndroMed diluent had a significant difference in increasing progressive motility, IAM, and IPM of Bali cattle spermatozoa at each stage of semen freezing, including post-dilution, post-equilibration, and post thawing when compared with controls. Based on the results, it can be concluded that adding 1 mmol of glutathione to the AndroMed diluent enhanced the quality and integrity of Bali cattle semen, including progressive motility, IAM, and IPM during the freezing process.

Correlation between in vitro sperm kinetic, oxidative stress assessments and field fertility of cryopreserved bull semen

This study assessed kinetic parameters and oxidative stress in bull sperm after post-thaw (PT) or after sperm selection by Percoll™ gradient, and thermo resistance test (SS + TRT) to identify useful indicators of field fertility. For the experiment, commercial doses of frozen semen were obtained from six Aberdeen Angus bulls. Three of the bulls were classified as high fertility and three as low fertility according to the IFert™ index provided by the international breeding company CRV Lagoa. Pooled semen samples were distributed between two treatment groups for analysis: post-thaw (PT) or sperm selection (SS) (Percoll™) and thermal resistance test (SS + TRT). The samples were evaluated using sperm kinetics (CASA) (motility %, progressive motility %, VCL µm/s, VSL µm/s, VAP µm/s, LIN %, STR % and WOB%), production of reactive oxygen species (ROS), lipid peroxidation, superoxide dismutase (SOD) enzyme activity and total antioxidant capacity. Data were analyzed using Two-Way ANOVA, considering the fertility index, the treatment used in the samples as effects, and the interaction between these factors. When a significant effect was observed, the values were compared using the Bonferroni test. A Pearson Correlation analysis was performed between the fertility indices and the sperm parameters analyzed in vitro, to evaluate the relationship between sperm quality and the fecundity rate obtained by the bulls. Sperm kinetic parameters, including total motility, progressive motile, and beat cross-frequency, were higher in low fertility compared to high fertility bulls (P &lt; 0.05). However, curvilinear velocity was greater in high fertility bulls followed by SS + TRT. Straight-line velocity, average path velocity, linearity, and beat cross-frequency beat were higher in high fertility bulls after SS + TRT. Reactive oxygen species was correlated with fertility after SS. In addition, there was a decrease in lipid peroxidation was observed only in high fertility bulls. However, lipid peroxidation and high fertility were correlated after PT and SS + TRT. The combination of in vitro sperm kinetic parameters predicted in vivo fertility more accurately than individual kinetic parameters. The lipid peroxidation of sperm is an important indicator of fertility in bulls. High fertility bulls appeared to be more susceptible to lipid peroxidation, which was only reduced in high fertility bulls, suggesting that their sperm can repair the damage induced by oxidative stress.