Sperm Cryopreservation Protocols Research Articles

Effect of cooling rates and equilibration times on post-thaw sperm quality of Kail rams

Context The conflicting findings regarding the impact of equilibration time on post-thawed sperm quality underscore the need for further research to evaluate the impact of equilibration time and cooling rate on post-thaw sperm quality of ram semen. Aims The current study aimed to assess the combined impact of cooling rates and pre-freezing equilibration times on post-thaw sperm quality in Kail ram semen (n = 5). Methods Semen collection was performed using an artificial vagina at 42°C. The pooled semen was divided into equal aliquots and subjected to either slow cooling (SC, −0.27°C/min) or moderate cooling (MC, −0.36°C/min) rates, transitioning from 37°C to 4°C. Equilibration times of 0, 4, 8, and 12 h were employed before freezing. Key results Semen samples undergoing the SC rate and equilibrated for 4 h exhibited higher (P < 0.05) percentages of progressive motile (PM), rapid progressive (RP), and medium progressive (MP) sperm compared with the MC rate. However, total motility remained unaffected by the cooling rate (P < 0.05). Semen equilibrated for 4 h demonstrated higher (P < 0.05) percentages of PM and RP sperm, as well as improved kinematics (curvilinear velocity, average path velocity, and straight-line velocity) compared with other equilibration times. Nevertheless, equilibration time had no (P > 0.05) impact on the amplitude of the lateral head displacement for semen samples subjected to the MC rate. Notably, the cooling rate did not affect post-thaw sperm kinematics, plasma membrane integrity, or live-sperm percentage (P > 0.05). Semen samples equilibrated for 4, 8, and 12 h exhibited a higher (P < 0.05) percentage of sperm with intact plasma membrane and viability than did those equilibrated for 0 h. Conclusions In conclusion, slow cooling rate and a 4 h equilibration period were shown to be optimal for preserving post-thaw sperm quality in Kail rams. Implications The findings highlighted the combined effect of cooling rate and equilibration time on post-thaw sperm quality for optimising sperm cryopreservation protocols in the context of ram semen.

Cryopreservation of goat spermatozoa in the Mekong Delta region of Vietnam: evaluating cryoprotectant effectiveness

Conserving and improving the quality of breeds is crucial for enhancing small ruminant production in Vietnam. To support this purpose, cryopreservation was offered as a promising method for semen preservation and maintaining genetic diversity. Moreover, many small-scale farmers in Mekong Delta rely on goats for both income and nutrition, making them an important part of farming activities. This study aimed to develop a sperm cryopreservation protocol and identify the optimal cryoprotectant for goat sperm in the Mekong Delta region of Vietnam. Semen samples were diluted with TCG-EggYolk freezing medium containing cryoprotectant agent (CPA) in a 1:1 ratio. The samples were then placed in 0.5 mL French straws, cooled to 15 °C and 5 °C, and subjected to nitrogen vapor before being immersed in liquid nitrogen at -196 °C. Experiment 1 utilized Glycerol as the CPA in concentrations of 0 %, 5 %, 8 %, and 11 %. Experiment 2 employed dimethyl sulfoxide (DMSO) as the CPA with the same concentrations. Thawed samples were evaluated for sperm quality. Results indicated that in Experiment 1, increasing the Glycerol concentration improved the overall motility, viability, and membrane integrity of sperm. The best outcomes were achieved with 8 % Glycerol, resulting in progressive motility of 57.5 % (P<0.05). In Experiment 2, the extender containing 8 % DMSO demonstrated significantly better performance in terms of overall motility, viability, and membrane integrity compared to 5 % or 11 % DMSO. Progressive motility was 53.5 % (P<0.05). In conclusion, the study determined that using 8 % Glycerol or 8 % DMSO is the optimal choice for freezing goat sperm in the Mekong Delta region of Vietnam.

Supplementation of Extender with Melatonin Improves the Motility, Mitochondrial Membrane Potential, and Fertilization Ability of Cryopreserved Brown-Marbled Grouper Sperm.

Sperm cryopreservation is a valuable tool for breeding, conservation, and genetic improvement in aquatic resources, while oxidative damage will cause a decline in sperm quality during this progress. Melatonin (MT), a natural antioxidant hormone, is used as an additive in sperm cryopreservation to reduce cellular damage from oxidative stress. Here, we aimed to investigate the effect of adding MT to the freezing medium in sperm cryopreservation of brown-marbled grouper (Epinephelus fuscoguttatus). Different concentrations of MT (0, 0.1, 0.25, and 0.5 mg/mL) were tested. We evaluated sperm motility, viability, apoptosis, mitochondrial membrane potential (MMP), and fertilization ability to assess the effects of MT supplementation. Our results demonstrated that the addition of MT to the extender improved the post-thaw motility, MMP, and fertilization ability of brown-marbled grouper sperm. The total motility, curvilinear velocity, straight linear velocity, and average path velocity in MT-treated groups (0.1 and 0.25 mg/mL) exhibited significantly higher values than that of the control group. A higher MMP (p < 0.05) was observed in the group treated with 0.25 mg/mL MT, suggesting that supplementation of MT in the extender might be able to protect mitochondrial membrane integrity effectively. Regarding fertilizing ability, 0.25 mg/mL MT yielded a significantly higher hatching rate than the control. An adverse effect was found with the concentration of MT up to 0.5 mg/mL, suggesting the possible toxicity of a high-dose addition. In this study, we optimized the sperm cryopreservation protocol of brown-marbled grouper, which might be valuable for sperm cryopreservation and sample commercialization of groupers and other fish.

Optimizing aquaculture-scale common carp artificial reproduction: a novel approach to sperm cryopreservation using large-volume containers and elevated thawing temperatures

IntroductionThe successful cryopreservation of common carp sperm is crucial for its application in aquaculture and selective breeding programs. This study investigates the efficacy of cryopreserving sperm in large containers (5 mL) with a low dilution rate (1:1) in three different cryoprotective media and thawing in different conditions.MethodsThe developed method utilizes a low-ionic (hypotonic) cryoprotective medium, freezing with a controlled cooling rate, and high-temperature sperm thawing (60°C). The investigation employs a detailed spermatozoon motility assessment.ResultsPost-thaw motility of 32.3% ± 14% and initial curvilinear velocity of 89 ± 20 μm/s across 30 males were observed. Principal component analysis of sperm kinematic characteristics revealed distinct populations of sperm cells exhibiting varying responses to cryopreservation. The developed method achieved successful fertilization comparable to that of the non-frozen control group using sperm from a single cryotube (2.5 mL, approximately 50 * 109 spermatozoa) to fertilize 200 g of eggs (1:120,000 egg:spz).DiscussionThis novel approach demonstrates an effective cryopreservation protocol for common carp sperm in large-volume cryo-containers in combination with low-ionic cryomedia and high thawing temperature, providing methods well-suited for fisheries practices and selective breeding programs. Future studies of the biological properties of different sperm subpopulations in post-thaw sperm samples can contribute to a deeper understanding of sperm biology, improve cryopreservation techniques, and enhance the success rates of assisted reproductive technologies.

Results of testing the protocol for cryopreservation of bull sperm using a concentrated dilutent medium

In recent years, dairy farming has been actively developing in Russia in many directions, providing consumers with milk, dairy products, meat and other raw materials. Particularly relevant are the automation of the processes of raising cattle and the selection of the most economically profitable dairy and meat breeds, which is facilitated by the development of artificial insemination in this agricultural sector. The success of artificial insemination is determined by many factors, including the quality of sperm extenders used and the correctness of cryopreservation protocols. The purpose of this study is to test a protocol for cryopreservation of Holstein bull sperm. The ejaculate was collected, evaluated, and pre-prepared (centrifugation, dilution, cooling). The freezing process was carried out using the method presented in the article by AnsariM.S., RakhaB. A. et al. (2015) for buffalo sperm: the straws were kept above liquid nitrogen vapor at a distance of 5 cm for 10 minutes, and then immersed in a Dewar flask. Sperm motility was assessed 3 times: after sperm collection, after cooling, and 0 hours after thawing. The negative impact of the cryopreservation process on the morphofunctional characteristics of sperm was determined. The study noted a decrease in the motility of progressively moving sperm by 21.53%, as well as a sharp increase in immobile sperm before and after cryopreservation from 6.8% to 33.82%. The number of nonprogressive spermatozoa was reduced from 9.12% to 2.91%. Changes in the morphological parameters of sperm as a result of cryopreservation (after cooling and 0 hours after thawing): the number of normal sperm decreased by 12%, the number of sperm with tail defects increased by 11% and head by 2%. Based on the data obtained, we can conclude that the use of this cryopreservation protocol, with preliminary preparation, helps preserve sperm viability. Further research will be aimed at studying and creating cryoprotectors that increase the safety of sperm of breeding bulls during deep freezing.

Qualitative indicators of sperm from producing goats before and after cryopreservation

Currently, artificial insemination, as one of the types of assisted reproductive technologies, is widely used in dairy and beef cattle breeding. The same cannot be said about such a promising direction in the agricultural industry as goat breeding. One of the limiting factors is the negative impact of low temperature on the morphofunctional charateristics of sperm of breeding goats. The purpose of this research was to test a protocol of sperm cryopreservation of stud goats with modification of prepreparation and subsequent assessment of the quality indicators of sperm before and after its deep freezing. A comprehensive assessment of sperm quality (volume, concentration, morphology, motility) of goats (n=10) was carried out using generally accepted methods and protocols. The assessment of sperm quality indicators included five stages: after sperm collection, two hours after cooling, after thawing: 0 hours, 1 and 2 hours. According on the obtained results, the sperm of breeding goats ha low cryoresistance. After cryopreservation (0, 1 and 2 hours after thawing), there is an increase in the number of sperm with tail damage by 7.5% (p≤0.05), 15.5% and 21.8% (p≤0.01), and also a decrease in the number of progressively moving sperm by 1.4; 1.6 and 2.5 times (p≤0.01) compared with the results of the assessment 0 hours after collection. The use of a deep two-phase sperm freezing protocol allows maintaining the viability of sperm with a progression of movements equal to 54.2±5.1% and a number of morphologically normal sperm equal to 64.1±1.9%. In this case, the prepreparation of sperm for the cryopreservation process (current protocol) includes sperm centrifugation (mode: 7000 rpm for 15 minutes), removal of seminal plasma, dilution 1:4 (OptiXcell diluent), cooling (4 hours at 4℃); sperm cryopreservation protocol: 1. immersion of goblets with paillettes 4 cm above liquid nitrogen for 7 minutes; 2. complete immersion in liquid nitrogen.

Transitioning from a research protocol to a scalable applied pathway for Xenopus laevis sperm cryopreservation at a national stock center: The effect of cryoprotectants.

Sperm cryopreservation is a critical tool for safeguarding and managing valuable genetic resources. Protocols for cryopreservation of Xenopus laevis sperm were available but lacking sperm quality evaluation and scalability and the outcomes were inconsistent. The goal of this study was to begin developing a center-level cryopreservation pathway for this species by integrating French straws as containers that would facilitate germplasm repository development. The objectives were to analyze the effect of: (1) three sperm concentrations (33, 50, and 100 × 106 sperm/mL) on post-thaw fertilization, (2) three final concentrations (2.5%, 5%, and 10%) of dimethyl sulfoxide, methanol, and dimethylformamide (DMFA) on sperm membrane integrity of fresh and frozen samples, (3) two concentrations (5% and 10%) of DMFA with and without 5% sucrose at four cooling rates (5, 10, 20, and 40°C/min) on sperm membrane integrity and motility, and (4) egg exposure to different concentrations of DMFA on fertilization. Few differences in sperm viability were found among fresh samples incubated in cryoprotectants, but thawed samples frozen in methanol or DMFA presented higher membrane integrity. Samples frozen in 10%DMFA at 20°C/min showed higher membrane integrity (60 ± 7%) than other DMFA concentrations and cooling rates, and the same total motility (30 ± 7%) as at 10°C/min. Higher DMFA concentrations (10%-13%) were detrimental for embryo development compared to lower concentrations (<6%). This study provided a reliable protocol for sperm cryopreservation in Xenopus laevis to yield an application pathway with potential for high throughput that can be used as a roadmap for work with other species.

Comparative characteristics of protocols for cryopreservation of ram semen

Purpose: study and comparison of protocols for cryopreservation of sperm of stud rams.Every day the popularity of sheep farming in Russia is increasing, since sheep are unpretentious in maintenance and at the same time they are multiproduct animals, providing the population with the necessary raw materials (wool, felt) and food products (meat, milk). For this reason, artificial insemination is of fundamental importance in the development of this branch of livestock farming, an important part of which is the correct cryopreservation of ram sperm. There are quite a few advantages of using cryopreserved sperm. Cryopreservation of sheep sperm contributes to the spread of the use of artificial insemination in this branch of the livestock industry. Freezing technology largely ensures the preservation of genetic material and its transportation, eliminating geographical barriers in the use of artificial insemination, and allows increasing the load on the ram sire without risk to his reproductive health. However, during the process of cryopreservation, sheep sperm receives irreversible damage due to cold shock, osmotic stress and oxidative processes, which lead to a decrease in the ability to fertilize sperm. Thus, chromatin may be damaged, membrane permeability increased, hyperoxidation and the formation of reactive oxygen species are possible, which affects the ability to fertilize. As a result, frozen ram semen may have low fertility. For this reason, it is extremely important to select an up-to-date freezing and thawing protocol with maximum recovery of viable and functional sperm of a given animal species for successful further use in artificial insemination. Technological advances in various fields of biotechnology have led to improvements in protocols, methods and equipment used in the laboratory, which have a positive impact on the reliability, accuracy of the procedure. This article summarizes research on the effects of different cryopreservation procedures, cooling rates, cooling times and thawing protocols on ram semen quality. In addition, a correct protocol for cryopreservation of Romanov breed sperm was formed, based on the results of our own research.

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis.

The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.

Cryopreservation of black seabream (Acanthopagrus schlegelii) sperm

In recent years, biotechnology has had a significant impact on the aquaculture industry, particularly in the field of breeding. Molecular selection breeding has emerged as a novel approach to breeding. Reducing the cost of genetic information for individuals with desirable traits after breeding has become an important research direction. Cryopreservation technology allows bypassing time and space constraints in genetic breeding, simplifying broodstock management. This study presents a detailed cryopreservation method for black seabream sperm, evaluating extender type, glucose concentration, cryoprotectant type and concentration, sperm-dilution ratio, and cooling protocols. Sperm motility parameters were analyzed using computer-assisted sperm analysis (CASA) before and after two days of freezing. This involved using an RS solution with a glucose concentration of 15 g/L and adding a 5% final concentration of EG as the sperm cryoprotectant. After mixing the sperm and solution at a ratio of 1:2, we subjected it to 5 min fumigation at 5 cm above the liquid nitrogen surface before plunging it into the nitrogen. Sperm motility reached 85.46 ± 7.32% after two days. Various enzymatic activities showed changes over 20 days post-cryopreservation. This improved cryopreservation protocol for black seabream sperm is beneficial for genetic breeding and reproduction and provides reference for studying the cryodamage mechanisms of black seabream sperm.

Cryopreservation of local black rabbit spermatozoa in the Mekong delta region of Vietnam: Evaluating cryoprotectant effectiveness.

Rabbit production is well developed in the Mekong Delta and Vietnam, with increased consumption of rabbit meat in both urban and rural areas. Consequently, meat rabbit breeds have become popular in almost all provinces in Vietnam, resulting in a decline in the population of Local Black rabbits over the years. To support breeding programs, there is a crucial need to develop a cryopreservation method. The objective of this study was to develop sperm cryopreservation protocols and determine the optimal cryoprotectant for Local Black rabbit spermatozoa. Rabbit spermatozoa were obtained and diluted with TCG-EggYolk freezing medium containing 5%, 8%, and 10% glycerol or DMSO in a 1:1 ratio. Glycerol and DMSO-free mediums served as the controls. Samples were put into 0.5-mL straws, cooled to 15°C and 5°C, then placed on nitrogen vapour for 15 min and directly immersed in liquid nitrogen at -196°C. Samples were thawed at 37°C for 60 s after being frozen for 72 h Sperm quality measures, such as viability, motility, and membrane integrity, were assessed both before and after freezing. The results showed that the average viability, average motility, and average membrane integrity were 61.9%, 53.9%, and 42.9%, respectively, after thawing with 5% glycerol, which was higher than the two higher concentrations of glycerol (8% and 10%) (p < .05). Similarly, with 8% DMSO, the average viability, average motility, and membrane integrity were 59.2%, 51.0%, and 41.8%, respectively, which were higher than the two concentrations of DMSO (5% and 10%) (p < 0.05). Therefore, the cryopreservation of local black rabbit sperm with 5% glycerol or DMSO 8% resulted in high sperm quality, but considering the practical aspect of minimizing the volume of the preservative, a concentration of 5% glycerol may be the best option.

The effect of residual pellet volume on cryoprotectant concentration and post-thaw motility of stallion sperm

Standard protocols for cryopreservation of stallion sperm involve centrifugation to remove plasma and concentrate ejaculates. Typically, semen is diluted 1:1 or higher to a concentration of 50 to 100 × 106/ml and centrifuged on a cushion. Most supernatant and cushion is aspirated leaving 5-10% of the original volume of diluted semen along with the sperm pellet. Pellets are resuspended in freezing diluent containing cryoprotectant to 200 × 106 sperm/ml. Due to the extreme variability of sperm concentration between ejaculates, the volume of resuspended sperm pellet and supernatant may contribute significantly to the final volume of the extended semen resulting in variability in the final percent of cryoprotectant (CPA) in the extended semen. To determine the degree of variability and the effect of the final CPA concentration on retention of sperm motility we determined final CPA concentration in extended semen of 129 ejaculates from 16 different stallions. Our hypothesis was that ejaculates cryopreserved with CPA concentration significantly lower than that present in the freezing diluent would have lower cryosurvival. Pellet volume was determined by weight and used to calculate the final CPA concentration prior to freezing for each ejaculate. The final CPA concentration was expressed as percentage of the CPA concentration in the original freezing extender. For the 129 ejaculates, the mean final CPA concentration aspercentage of original was 69.5%, median 73.7%, min 39.2% and max 87.8%. Final CPA concentration 1.3 - 5.1%. Sperm motility was analyzed by CASA for fresh extended and post-thaw samples. Thawed samples were diluted in centrifugation media and incubated (37C for 30 minutes) prior to analysis. For each sample a minimum of 5 fields and 400 motile sperm were analyzed (40 frames at 60 frames/sec); progressive cells defined as (VAP > 50 mic/sec and STR > 75%). Cryosurvival was expressed as percentage change in motility between pre-freeze and post-thaw values. Ejaculates were divided into 5 groups based on final CPA concentration as percentage of the original extender amount (G1 = 40-50%, G2 = 50-60%, G3 = 60-70%, G4 = 70-80%, G5 = 80-90%). Mean values for change in motility were compared between groups using ANOVA with multiple comparisons. Decrease in percent total motility (mean,sd) was less (p<0.05) in G5 (22.8,11.0) compared to all other groups G4 (31.3,11.3), G3 (31.2,13.1), G2 (29.5,8.8), G1 (30.9,10.8). Decrease in progressive motility was less (p<0.05) in G4 (8.5,14.8) and G5 (2.7,10.5) vs G1(17.8,8.7), G5 vs G2 (13.6,10.9) and G5 vs G3 (12.9,14.3). These data suggest that current protocols for cryopreservation of stallion semen may result in inadequate CPA concentration in extended semen of many ejaculates. Further experiments are underway to determine critical thresholds.

Optimization of sperm cryopreservation protocol for Basa catfish (Pangasius bocourti)

The aim of this study was to optimize a sperm cryopreservation protocol for Basa catfish (Pangasius bocourti). Key factors for the efficiency of cryopreservation, including cryoprotectants, thawing conditions, equilibration times, dilution ratios and freezing methods, were investigated. The suitable time for post-thaw storage as well as pre-freezing cool storage was also examined. Five cryoprotectants (dimethyl sulfoxide, Me2SO; dimethylformamide, DMF; ethylene glycol, EG; propylene glycol, PG; N-methylacetamide, MA) at different final concentrations (5, 10 and 15%) were tested, 10% PG provided the best cryoprotective effect. Thawing temperature at 30–40 °C yielded significantly higher post-thaw motility than 20, 25, 50 or 60 °C. No obvious effect on sperm motility was detected either in equilibrated or thawed samples during a 2-h equilibration. Regarding dilution ratio (semen/cryomedium, v/v), percentage of motile spermatozoa was significantly higher at ratios of 2:1, 1:1 and 1:3 than those with higher ratios (1:5, 1:7 and 1:9). Thawed sperm was sensitive to post-thaw storage, but no reduction in motility was detected within 30 min. Further evaluation of the effective pre-freezing storage time indicated that sperm in diluted form had more advantage in maintaining its freezability, which could be chilled for 24 h before freezing without compromising post-thaw sperm motility. P. bocourti sperm could be successfully cryopreserved with both a programmable freezer and the floating frame technique (frozen 5 or 7 cm above the surface of liquid nitrogen). Cryopreserved sperm (77.5 ± 5.1%) fertilization was not significantly different from fresh sperm (80.9 ± 4.7%) at the ratio of 2 × 105 spermatozoa per egg. Our results provided more detailed suitable conditions for P. bocourti sperm cryopreservation than previous studies. Standardizing the cryopreservation protocol and storage time would be helpful in facilitating artificial reproduction in this species.

Sperm cryopreservation does not affect live birth rate in normozoospermic men: analysis of 7969 oocyte donation cycles.

Does sperm cryopreservation influence the reproductive outcomes of normozoospermic patients in oocyte donation cycles? After controlling for confounders, the use of cryopreserved semen from normozoospermic patients does not affect pregnancy and live birth rates after elective ICSI. Sperm cryopreservation by slow freezing is a common practice in ART. While frozen-thawed semen typically presents reduced motility and vitality, its use for ICSI is generally considered adequate in terms of reproductive outcomes. Nevertheless, most studies comparing reproductive outcomes between fresh and cryopreserved sperm include patients with severe male factor (testicular sperm, oligo-, and/or asthenozoospermia) or women of advanced maternal age, where the altered quality of the gametes can partially mask the full effect of freezing/thawing. The study included a retrospective cohort of 7969 couples undergoing their first oocyte donation cycle between January 2013 and December 2019 in one large clinic, using normozoospermic semen from the male partner. All cycles involved elective ICSI, fresh oocytes, and a fresh embryo transfer, either at cleavage or blastocyst stage. Two study groups were established based on the sperm status: fresh (n = 2865) and cryopreserved (n = 5104). A slow freezing protocol was used for all sperm cryopreservation. Sperm washing, capacitation, and selection prior to ICSI were performed identically for fresh and frozen-thawed samples, using pellet swim-up. Fertilization rate (FR), pregnancy (biochemical and ongoing), and live birth rates were compared between study groups using univariate and multivariate regression analyses. Male and female age, sperm concentration and motility after ejaculation, and number of oocytes inseminated were similar between cycles using fresh or cryopreserved sperm. Analysis by Student's t-test did not indicate a significant difference in FR between fresh and cryopreserved sperm (P = 0.0591); however, after adjusting for confounders, this difference reached statistical significance: 74.65% FR for fresh (CI 95%: 73.92-75.38) versus 73.66% for cryopreserved sperm (CI 95%: 73.11-74.20), P = 0.0334. The adjusted regression analysis revealed higher odds of biochemical pregnancy when using fresh sperm (odds ratio (OR): 1.143, P = 0.0175), but no significant effects of sperm cryopreservation were observed for ongoing pregnancy (OR: 1.101, P = 0.0983) and live birth (OR: 1.082, P = 0.1805). Caution should be exerted when extrapolating these results to different protocols for sperm cryopreservation and selection, or to IVM, advanced maternal age and classical IVF cycles, which were excluded from analysis. Owing to the retrospective nature of the study, some uncontrolled for variables may affect the results. Sperm cryopreservation does not affect pregnancy and live birth rates in normozoospermic patients, and although it may lower FR s slightly, this would not be clinically relevant. In line with previous studies that included patients with an apparent male or female factor, sperm cryopreservation is a safe and convenient technique. The study received no external funding and all authors have no conflicts of interest to declare. N/A.

Applying sperm collection and cryopreservation protocols developed in a model amphibian to three threatened anuran species targeted for biobanking management

The challenge of replicating environmental cues in amphibian captive assurance colonies has been shown to be one of the contributing factors leading to limited breeding success. To circumvent this challenge, assisted reproductive technologies (ART) have been developed in model organisms and can now be applied to at-risk species. This study tested the transferability of cryopreservation protocols developed in the Fowler's toad (Anaxyrus fowleri) to three threatened anurans, the Chiricahua leopard frog (Lithobates chiricahuensis), Puerto Rican crested toad (Peltophryne lemur), and Houston toad (Anaxyrus houstonensis). Males were administered human chorionic gonadotropin and/or gonadotropin releasing hormone-analog to elicit spermiation, and spermic urine was collected and analyzed for motility. Samples were cryopreserved with 10 % N,N-dimethylformamide + 10 % trehalose + 0.25 % bovine serum albumin and frozen at two rates, −32 to −45 °C/min and − 20 to −29 °C/min. Results show that sperm frozen at −32 to −45 °C/min recovered significantly higher post-thaw sperm motility than −20 to −29 °C/min for P. lemur (p = 0.04) and L. chiricahuensis (p = 0.03). When compared across species, L. chiricahuensis had significantly higher post-thaw motility at both freezing rates (p < 0.05) than the other species, yet there was no difference between sperm motility between the three bufonid species for either freezing rate (p > 0.06). These results indicate that transferability of a cryopreservation protocol between species is possible, and differences in sensitivity and response to cryopreservation may exist between genera.

Protocol for common carp sperm cryopreservation for aquaculture-scale artificial reproduction

Protocol for common carp sperm cryopreservation for aquaculture-scale artificial reproduction

Optimized protocols for sperm cryopreservation and in vitro fertilization in the rat.

Laboratory rats have been used in biomedical research for over 170 years. Recently, genome editing technology has facilitated the generation of genetically modified rats worldwide. This development has increased the demand for efficient preservation and production of rat resources. Sperm cryopreservation is the most efficient and robust means to archive genetic resources, and this technique reduces the number of animals required for colony management. Previously, we have reported a protocol for rat sperm cryopreservation and in vitro fertilization using frozen-thawed sperm. Here we describe an improved in vitro fertilization protocol to enhance the fertilization rate of cryopreserved sperm in major strains of rats. In this optimized protocol, treatment of frozen-thawed rat sperm with a high concentration of bovine serum albumin (40 mg/ml) results in a high in vitro fertilization rate. This protocol consists of three main steps: preparation of cryopreserved sperm, in vitro fertilization using cryopreserved sperm and embryo transfer. This process takes approximately 1 month to produce live pups from cryopreserved sperm. This protocol can be easily implemented by researchers and technicians with experience in reproductive engineering technology; it can also be used, albeit with some practice, by researchers and technicians who have no experience in reproductive techniques. This sperm cryopreservation and in vitro fertilization protocol for rats will provide an efficient system for the archiving and production of genetically modified rats for the transgenic community.

Melatonin Supplementation for the Cryopreservation of Canine Sperm.

Antioxidants can be used in sperm cryopreservation protocols to reduce oxidative stress that occurs due to the cryopreservation process. The aim of this study was to evaluate the effects of melatonin supplementation on quality and oxidative stress parameters in cryopreserved canine sperm. Eighteen sperm ejaculates were collected from 6 Frenchie Bulldog males (3 collections per male). Sperm motility parameters, membrane integrity, and sperm morphology were analyzed before the cryopreservation process. The extender used in cryopreservation was composed of Tris-egg yolk and ethylene glycol 5% was added as a cryoprotectant. The cryoprotective medium was supplemented with 1.0, 1.5, 2.0, 2.5, and 3.0 mM melatonin, and the control group (without melatonin). Post-thaw sperm was evaluated as described for fresh sperm and oxidative stress parameters (lipid peroxidation, catalase, and superoxide dismutase). Post-thaw sperm motility parameters, membrane integrity, sperm morphology, and oxidative stress parameters did not differ (p > 0.05) among the control group and samples supplemented with melatonin. The results of this study showed that melatonin supplementation had no positive or negative effect on the parameters evaluated. Thus, it is suggested that different concentrations of melatonin be tested to assess its effectiveness as an antioxidant in the cryopreservation process in canine sperm.

Cryopreservation of Roughscale Sole (Clidoderma asperrimum) Sperm: Effects of Cryoprotectant, Diluent, Dilution Ratio, and Thawing Temperature.

Simple SummaryThe roughscale sole, Clidoderma asperrimum, is found in the East and West Seas of Korea, waters north of Hokkaido in Japan, as well as the East China Sea, the East Pacific, and the Canadian Maritimes. In 2021, this fish was categorized as an endangered species. Thus, there is a need to maintain gametes by freezing sperm. This study investigated the impacts of the cryoprotective agent, diluent, dilution ratio, and thawing temperature on the cryopreservation of fish sperm. In this investigation, sperm dilution 1:1 with a mixture of 10% dimethyl sulfoxide + Stein’s solution and thawing at 10 °C provided the most effective DNA damage prevention. These results support the development of a roughscale sole sperm cryopreservation procedure.The roughscale sole, Clidoderma asperrimum is categorized as an endangered species. Sperm freezing is essential for preserving gametes. This study examined the CPA concentration, diluent, dilution ratio, and thawing temperature to design a sperm cryopreservation protocol for roughscale sole. The variables examined included sperm motility and kinematics, cell survival, fertilization, and DNA fragmentation. Sperm motility parameters were assessed via computer-assisted sperm analysis using a CEROS II instrument. Cell survival rate and DNA damage were assessed using the Cell Counting Kit-8 and single-cell gel electrophoresis assay, respectively. Sperm preservation was tested using several CPAs, including ethylene glycol, dimethyl sulfoxide (DMSO), glycerol, propylene glycol, and methanol. The diluents tested were 300 mM sucrose, 300 mM glucose, Stein’s solution, Ringer’s solution, and Hank’s solution. The optimal conditions for sperm cryopreservation were 10% DMSO + Stein’s solution. After thawing, sperm motility was highest with a 1:1 dilution ratio (sperm to CPA + diluent), at 69.20 ± 0.32%; thawing at 10 °C was optimal for post-thaw motility (72.03 ± 0.95%). The highest fertilization rate (40.00 ± 1.22%) was obtained using DMSO. The fresh sperm had the lowest tail DNA, followed by 10% DMSO + Stein’s solution. The developed cryopreservation methods can be used in roughscale sole hatcheries.

Post-Thaw Sperm Recovery Rates; A Retrospective Study

Background: Cryopreservation is currently the only fertility preservation option available to patients undergoing gonadotoxic treatment. Much research in the field focusses on techniques, media and equipment employed to cryopreserve cells, and demonstrating cell viability after using such. Comparatively, there is a death of information available on the procedures and equipment used to thaw cryopreserved cells, or estimates for post-thaw recovery. Aim: This is a retrospective study assessing semen parameters in post-thawed specimen from patients who attended the Royal Women’s Hospital Andrology Unit for sperm cryopreservation prior to gonadotoxic or other treatments. The study aims to examine the relationship between sperm cryopreservation technique, media and period of storage with post-thaw sperm parameters. Method: Patients with written consent to discard their stored semen were selected for inclusion in the study (n=59); samples with sperm concentration [Formula: see text]1 million/ml prior to freezing (n=11) were excluded. Semen samples were thawed accordingly: 5 minutes at room temperature followed by incubation at 37[Formula: see text]C for sperm straws; vials were transferred directly to a mixer incubator at 37[Formula: see text]C. All samples were immediately assessed for sperm concentration and motility. Recovery rates were calculated and data were analysed using SPSS (IBM, USA) version 25. Results: Progressive motility recovery was 32.4% ± 20.6. Patients were divided into groups according to period of storage, and showed no significant differences for recovery of motility and concentration (p=0.44, 0.96, respectively). Furthermore, progressive motility recovery rates were not significantly different between freezing techniques (p=0.116) and media (p=0.099). Conclusion: Data from this study are preliminary and further study is warranted to fully extrapolate the relationship between sperm cryopreservation protocols and period of storage on recovery rates. The lack of relationship between these factors exhibited in the data could suggest that research and development in thaw technologies may be more pertinent to improving sperm recovery rates.