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Cryopreservation facilitates long-term semen storage and enables the exchange of genetic material among elite livestock over extensive distances. A decrease in sperm quality is an unavoidable outcome of the cryopreservation process. Prior research has established that incorporating cryoprotectants into the diluent can mitigate freeze-induced damage and enhance sperm quality. This study aims to assess the impact of Epimedium polysaccharide (EPS) on the cryopreservation of goat semen. Samples were obtained from six healthy goats following an initial examination. One portion of the semen was diluted with a base solution containing EPS for treatment purposes, whereas another was diluted without EPS, serving as the control. Results indicated that varying concentrations (1, 2, 3, 4, 5 mg/mL) of EPS in the diluent enhanced both physiological characteristics and antioxidant enzyme activities in cryopreserved goat sperm. Further analysis showed that the 3 mg/mL EPS concentration significantly improved sperm motility (52.10 %), plasma membrane integrity (57.01 %), tail plasma membrane integrity (52.37 %), acrosome integrity rate (52.45 %), and antioxidant enzyme activities relative to other groups (P < 0.05). Additionally, the inclusion of 3 mg/mL EPS substantially improved the sperm's fertilization capability. In conclusion, our experiments confirm that EPS supplementation significantly enhances sperm quality post-freezing, with 3 mg/mL identified as the optimal concentration. [ABSTRACT FROM AUTHOR]

Oocyte cryopreservation is increasingly being used in reproductive technologies for conservation and breeding purposes. Further development of oocyte cryopreservation techniques requires interdisciplinary insights in the underlying principles of cryopreservation. This review aims to serve this purpose by: (1) highlighting that preservation strategies can be rationally designed, (2) presenting mechanistic insights in volume and osmotic stress responses associated with CPA loading strategies and cooling, and (3) giving a comprehensive listing of oocyte specific biophysical membrane characteristics and commonly used permeation model equations. It is shown how transport models can be used to simulate the behavior of oocytes during cryopreservation processing steps, i.e., during loading of cryoprotective agents (CPAs), cooling with freezing as well as vitrification, warming and CPA unloading. More specifically, using defined cellular and membrane characteristics, the responses of oocytes during CPA (un)loading were simulated in terms of temperature- and CPA type-and-concentration-dependent changes in cell volume and intracellular solute concentration. In addition, in order to determine the optimal cooling rate for slow programmable cooling cryopreservation, the freezing-induced cell volume response was simulated at various cooling rates to estimate rates with tolerable limits. For vitrification, special emphasis was on prediction of the timing of reaching osmotic tolerance limits during CPA exposure, and the need to use step-wise CPA addition/removal protocols. In conclusion, we present simulations and schematic illustrations that explain the timing of events during slow cooling cryopreservation as well as vitrification, important for rationally designing protocols taking into account how different CPA types, concentrations and temperatures affect the oocyte. • Membrane permeability determines the cell volume response upon osmotic stress. • Subzero membrane permeability to water determines freezing-induced dehydration. • Simulations reveal the timing and concentrations needed for step-wise CPA loading. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation technology significantly contributes to the safeguarding of genetic resources, particularly for endangered species, and supports the use of artificial insemination in domestic animals. Therefore, cryopreservation can negatively affect sperm health and function leading to reduce the freezing ability and fertility potential. Therefore, it is essential to prioritize the improvement of cryotolerance in cryopreserved sperm to enhance reproductive efficiency and ensure sustainability in livestock herds. The main reason for sperm dysfunction after thawing may be related to the excessive amount of oxidative stress (OS) produced during cryopreservation. Scientists have different ways for counteracting this OS including the use of plant extracts, enzymes, minerals, anti-freezing proteins, and amino acids. Recently, one such amino acid is L-proline (LP), which has multiple roles such as osmotic and OS defense, nitrogen, and carbon metabolism, as well as cell survival and signaling. LP has been found in seminal plasma and has recently been added to the freezing extender to improve the various post-thaw parameters of sperm. This improvement is related to the ability of LP to reduce the OS, sustain the plasma membrane and to act as an osmoregulatory agent. Moreover, LP can suppress cell apoptosis by modulating intracellular redox in sperm. This review addresses the ongoing research on the addition of L-proline as an osmoregulatory agent in freezing extenders to increase the cryotolerance of animal spermatozoa to freeze-thaw. [Display omitted] • L-proline (LP) improves the sperm motion after sperm cryopreservation in various animals. • This improvement is related to the ability of LP to reduce the OS, sustain the plasma membrane and osmoregulatory agent. • LP can suppress cell apoptosis by modulating intracellular redox in sperm [ABSTRACT FROM AUTHOR]

ProAKAP4 is identified within the flagellum of spermatozoa in various mammalian species, serving as a structural protein associated with motility parameters. This investigation focuses on the presence of proAKAP4 in donkey sperm, elucidating its localization, molecular characteristics, and its correlation with motility descriptors and mitochondrial membrane potential. Twelve ejaculates from Catalan donkeys were analyzed in this study. The initial steps involved proAKAP4 sequencing and detection through Western blotting and immunofluorescence. Post-thaw assessments were conducted at 0, 1, and 3 h, encompassing proAKAP4 levels, sperm motility analyzed via Computer-Assisted Sperm Analysis (CASA), and mitochondrial membrane potential determined by flow cytometry using the JC-1 stain. The findings reveal that proAKAP4 in donkeys exhibits a characteristic localization at the principal piece of the flagellum, consistent with observations in other mammals. The molecular weight of proAKAP4 is determined to be 100 kDa. Significantly, a positive correlation (p ≤ 0.05) is established between proAKAP4 concentration and both total and progressive motility. The presence of cryoprotectant is associated with a lower proAKAP4 concentration. Notably, proAKAP4 experiences a substantial decrease (p ≤ 0.05) during the initial hour post-thawing. In conclusion, proAKAP4 is identified in donkey sperm, akin to its presence in other mammals. It exhibits a positive correlation with total and progressive motility, its concentration is notably affected by the presence of cryoprotectant with significant consumption observed during the initial hour following thawing. These findings contribute to our understanding of proAKAP4 dynamics in donkey sperm, providing insights that may have implications for semen preservation and reproductive technologies in equids. • ProAKAP4 is labeled in donkeys like in other mammals. • In donkey, proAKAP4 correlates with total and progressive motility. • Upon thawing of the semen, higher proAKAP4 concentration was observed removing cryoprotectant [ABSTRACT FROM AUTHOR]

The aim of the present study was to establish a protocol for solid surface vitrification of peccary ovarian tissue by using different cryoprotectants. Ovarian pairs from five adult females were fragmented and two fragments (fresh control group) were immediately subjected to morphological evaluation using classical histology, transmission electron microscopy, and viability analysis using fluorescent probes. The remaining fragments (n = 18) were vitrified using a solid surface method with different concentrations (3 or 6 M) of ethylene glycol (EG), dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). After 2 weeks, samples were re-warmed and evaluated. A decrease in the percentage of morphologically normal preantral follicles (PFs) was verified for all the groups in comparison to the fresh control (92.0 ± 2.8%); however, if only the primordial follicles are considered, the most effective preservation (P < 0.05) was achieved with the use of EG at 3 M (74.2±7.3%) or DMSO at 6 M (75.0 ± 4.2%). Ultrastructural analysis indicated there were well-preserved PFs in all the groups evaluated, having well-defined membranes, a few vacuoles, and organelles that were uniformly distributed throughout the cytoplasm, mainly round and elongated mitochondria in close association with lipid droplets. Viability was preserved (P < 0.05) with the use of EG at 3 (97%) or 6 (97%) M, DMSO at 3 (100%), and DMF at 6 (97%) M. Solid surface vitrification, therefore, is an effective method for conservation of peccary female germplasm, especially with the use of EG at 3 M, which was highly effective for preservation of both the morphology and viability of PFs. [ABSTRACT FROM AUTHOR]

The aim of the present study was to compare the survival and developmental rate of canine isolated preantral follicles (PAFs) after cryopreservation with different methods (closed vs open vitrification). Follicles were isolated from ovaries randomly divided into three groups: fresh control, OPS (open pulled straw) vitrified and cryotube (CT) vitrified. Post-thaw viability of follicles and oocytes was assessed. Fresh and vitrified/thawed PAFs were cultured in 20 µl drops of FSH-supplemented medium for 10 days. Follicular growth, survival rate, estradiol production and ovulation rate were examined. CT method resulted in lower rate of live cells (58.7%) and oocytes (38.8%) than that of fresh ones (83.6% and 64%, respectively) and OPS (80.3% and 79.3%, respectively). Survival rate was similar to fresh follicles in OPS group (98.5% and 95.4%, respectively), while CT decreased the survival to 81.2%. Fresh follicles showed continuous growth, while CT follicles stopped to increase their size after 2 day. In the OPS vitrified follicles, this halting occurred between Day5 and Day10. Fresh follicles showed the highest estradiol production (range: 26.9 – 266.2 pg/ml). Comparing the two vitrified groups, lower estradiol concentration range was measured in the CT group (7.8–48.7 pg/ml vs. 15.4–89.6 pg/ml). Ovulation rate in each group was lowest in the OPS group (1.7% vs 7% and 8.9% in fesh and CT, respectively). Our data show that OPS vitrification provides superior survival rate, in vitro growth and hormonal production to CT. To our knowledge, these are the first results on comparing different cryopreservation protocols on canine isolated preantral follicles. • Canine preantral follicles were cryopreserved with open and closed vitrification. • Post-thaw survival of follicles and oocytes inside was better in open system. • In vitro growth rate and estradiol production was affected by the vitrification. • OPS follicles showed intensive growth and higher hormone production than that of CT. • OPS is a suitable method to preserve canine preantral follicles. [ABSTRACT FROM AUTHOR]

Glycerol is a cryoprotectant used widely for the cryopreservation of animal sperm, but it is linked to a decrease in fertility. The mechanism underlying the negative effects of glycerol remains unclear. Therefore, in this study, we aimed to gain a better understanding by using the chicken model. First, we investigated the impact of increasing the concentration of glycerol during insemination on hen fertility. Our findings revealed that 2% glycerol resulted in partial infertility, while 6% glycerol led to complete infertility. Subsequently, we examined the ability of sperm to colonize sperm storage tubules (SST) during in vivo insemination and in vitro incubation. The sperm used in the experiment were stained with Hoechst and contained 0, 2, or 6% glycerol. Furthermore, we conducted perivitelline membrane lysis tests and investigated sperm motility, mitochondrial function, ATP concentration, membrane integrity, and apoptosis after 60 min of incubation with different glycerol concentrations (0%, 1%, 2%, 6%, and 11%) at two temperatures to simulate pre-freezing (4 °C) and post-insemination (41 °C) conditions. Whereas 2% glycerol significantly reduced 50% of sperm containing SST, 6% glycerol completely inhibited SST colonization in vivo. On the other hand, in vitro incubation of sperm with SST revealed no effect of 2% glycerol, and 6% glycerol showed only a 17% reduction in sperm-filled SST. Moreover, glycerol reduced sperm-egg penetration rates and also affected sperm motility, bioenergetic metabolism, and cell death at 4 °C. These effects were observed when the concentration of glycerol exceeded 6%. Furthermore, at 41 °C, glycerol caused even greater damage, particularly in terms of reducing sperm motility. These data altogether reveal important effects of glycerol on sperm biology, sperm migration, SST colonization, and oocyte penetration. This suggests that glycerol plays a role in reducing fertility and presents opportunities for improving sperm cryopreservation. [Display omitted] • Glycerol provides excellent cryoprotection and shows cytotoxicity to sperm cells. • In hens, 2% or 6% glycerol added into semen induce partial or total infertility. • Glycerol affects sperm migration and storage in the oviduct. • Glycerol causes more negative modifications of sperm biology at 41 °C than at 4 °C. • Removing glycerol prior to insemination is the most valuable solution so far to avoid fertility reduction. [ABSTRACT FROM AUTHOR]

Highlights • E. maclovinus is a potentially farmable marine fish recently putted in captivity. • Cold-storage effect over sperm ultrastructure is observed by SEM. • Cold-storage effect over sperm quality is quantified. • Use of Cortland solution improves E. maclovinus sperm storage time. Abstract Patagonian blenny (E. maclovinus) is a marine species recently placed in captivity and which are potentially farmable. Understanding and improving its sperm capacity to withstand short-term storage conditions is a key element of initiating an artificial propagation program for this species. The aim of this study is to evaluate the ultrastructure and quality of E. maclovinus sperm during refrigerated storage. To address this objective, scanning electron microscopy (SEM), cytofluorimetric analysis (membrane integrity; reactive oxygen species generation; mitochondrial membrane potential) and cell respiration/mitochondrial-function analysis (ATP content; oxygen consumption) could be useful for optimizing or improving management for artificial reproduction of this species. Severe damage of plasma membranes was observed by SEM at day 7 and 14 of in vitro storage. Analyses of sperm quality were conducted during the 14-day cold storage period when sperm were in diluted (with Cortland solution) and undiluted conditions. When there were diluted conditions, there was greater preservation of motile capacity (from day-7; P < 0.05), membrane integrity (from day-7; P < 0.05), mitochondrial membrane potential (from day-10; P < 0.05) and ATP stores (from day-3; P < 0.05). Oxygen consumption indicators were 18.6% ±14.7% greater in the undiluted samples from day-3, and 32.1%±2.1% of the total spermatozoa had ample amounts of superoxide anion in both undiluted and diluted semen on day-0. The use of Cortland solution extended the viability of sperm when there were longer storage times. Factors that have a greater effect on the quality of semen during storage are reactive oxygen species generation and ATP depletion. In conclusion, Patagonian blenny spermatozoa can be stored at 4 °C between 7 and 10 days using Cortland solution. [ABSTRACT FROM AUTHOR]

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

The aim of this study was to assess different concentrations of sucrose-based extenders combined with bovine serum albumin (BSA) as an alternative to stallion sperm cryopreservation with permeable cryoprotectants. Semen samples ( n  = 16) were collected from six stallions. Sperm was cooled, filled in 0.5 mL straws and frozen in nitrogen vapor. Post-thaw sperm kinetic parameters, plasma and acrosome membrane integrity were statistically compared among treatments. In Experiment 1, extenders containing 1% of BSA and different concentrations of sucrose (mmol/L, M): 0, 50, 100, 250, 350 and 450 mM were compared. Use of sucrose [100 mM (S2)] resulted in greater values for most of the sperm kinetic parameters assessed ( P  < 0.001). There were no differences for plasma membrane integrity, except for when sucrose was used at 50 and 250 mM concentrations, and plasma membrane integrity was less ( P  < 0.05) when these concentrations were used than with the other sucrose concentrations. In Experiment 2, the selected sucrose extender (S2) was compared to an extender containing glycerol as permeable cryoprotectant. Use of the S2 extender resulted in a lesser proportion of sperm with denuded-acrosomes ( P  < 0.05) in comparison to use of glycerol and values for several kinetic parameters were also greater ( P  < 0.05) with use of S2. There were no significant differences for the other parameters assessed in this study. In conclusion, stallion sperm can be frozen in the absence of permeable cryoprotectants, using a combination of sucrose 100 mM with BSA-1% as alternative agents. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of different concentrations of sucrose combined with bovine serum albumin (BSA), as non-permeable cryoprotectants, on donkey sperm parameters after cryopreservation, in comparison to a control extender containing glycerol. Semen from five Andalusian donkeys (n = 12) were centrifuged and resuspended with a commercial extender for equine sperm (Gent A, Minitube) adding 1% BSA and different concentrations (M, mol/l) of water-diluted sucrose: 0.05, 0.1, 0.25, 0.35 and 0.45. Thereafter, semen (n = 24) were diluted in the same base extender containing 0.25 M sucrose (S25) or glycerol (GLY, Gent B). Sperm were slowly cooled, filled in 0.5 ml straws and frozen in nitrogen vapours. Post-thaw samples were assessed for sperm motility, plasma membrane and DNA integrity and results were compared by ANOVA. In Experiment 1, sperm motility was significantly higher ( P <  0.001) for S25 than the remaining treatments, and no differences were found for plasma membrane or DNA integrity. In Experiment 2, no differences were found between S25 or GLY for sperm motility and DNA integrity but plasma membrane integrity was significantly higher ( P  < 0.05) for S25. In conclusion, the extender with sucrose 0.25 M combined with BSA can be considered as an alternative to conventional extenders with glycerol for donkey sperm cryopreservation. [ABSTRACT FROM AUTHOR]

The aim of this study was to document the effects of cryopreservation on sperm motility and viability in Grey mullet Mugil cephalus . Cryopreservation of sperm was attempted by using two extenders ringer solution for marine fish (RSMF) and V2 extender (V2E) and cryoprotectants dimethylacetamide (DMA), dimethylsulfoxide (DMSO), ethylene glycol (EG), glycerol (GLY), propylene glycol (PG) and methanol (MeOH). Cryoprotectants were assessed at different concentrations individually as well as in combination with varying equilibration times (10 and 30 min). For optimization of freezing rate, four freezing protocols (−5, −10, −20 and −30 °C/min) were evaluated. After achieving final temperature, samples were plunged in liquid nitrogen (−196 °C) and stored for a week. Samples were subsequently thawed in a water bath at 30 °C for assessment of sperm motility and viability. Results indicated that cryomedium constituting of V2E extender + 10% glycerol with a dilution ratio of 1:1 (sperm: cryomedium) at an equilibration time of 5 to- 10 min and freezing rate of −20 °C/min was more desirable compared with other factors that were assessed. Use of this protocol resulted in retaining the greatest sperm motility grade 3.0 ± 0.0 (50%–80% sperm movement, fast swimming) and 48.19 ± 3.12% of sperm viability. The results of the present study, therefore, provide base-line data for establishing a protocol for sperm cryopreservation in M.cephalus . Further studies are, however, required for optimization of most suitable sperm cryopreservation protocol. [ABSTRACT FROM AUTHOR]

During the cryopreservation process, spermatozoa are exposed to hypertonic solutions contributed by the high concentration of cryoprotectant. During addition and removal of cryoprotectant the spermatozoa are subjected to a substantial osmotic stress. Spermatozoa of different species and different stages of maturation may have different susceptibility to osmotic stress depending on the biology of the cell membrane and this will affect their tolerance to the freezing-thawing stress. The aims of this study were to determine the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to study the effect of osmotic stress on the feline spermatozoa of different epididymal regions. Epididymal spermatozoa from three regions (caput, corpus and cauda) were pre-exposed to various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10 min and returned to 300 mOsm afterward. Percentage of motile spermatozoa was measured subjectively and membrane integrity (SYBR-14 positive cells) was evaluated prior to and after exposure to different osmolalities. The mitochondrial membrane potential (JC1) of spermatozoa were evaluated using flow cytometer and compared between epididymal regions (caput, corpus and cauda). All the parameters were compared using a mixed procedure. The percentage of motile epididymal spermatozoa decreased significantly when spermatozoa were exposed to 75 mOsm and 600 mOsm. Epididymal spermatozoa showed signs of damage when pre-exposed to 900 and 1200 mOsm and returned to isotonic condition as significant reduction of membrane integrity and mitochondrial membrane potential were observed (P < 0.05). The plasma membrane of spermatozoa from the cauda epididymal region showed higher susceptibility to osmotic stress than the other regions as demonstrated by a significant difference between regions after return to isotonicity from 900 mOsm (P > 0.01) and a difference between caput and corpus after return from 1200 mOsm (P < 0.05). The corpus and cauda epididymal spermatozoa had higher percentage of spermatozoa with high mitochondrial membrane potential than those from caput when exposed to 75, 300 and 600 mOsm (P < 0.05). In conclusion, a single step exposure to hypertonic solution of greater than 600 mOsm prior to return to isotonic condition can cause severe damage to sperm membrane and mitochondrial membrane potential compared to non-returning (exposure to various osmolality but not returned to isotonic condition). Changes in osmolality impacted mostly on sperm motility. Spermatozoa from cauda epididymis were more susceptible to osmotic stress compared to those from corpus and caput indicating that the maturation changes in the sperm membrane during passage through the epididymis increase susceptibility to the osmotic changes that may occur during sperm cryopreservation. [ABSTRACT FROM AUTHOR]

The aim of this study was to analyse the characteristics of ram spermatozoa subjected to varying concentrations of sucrose, and the influence of storage temperature (22 °C or 5 °C) prior to vitrification. Ejaculated semen was diluted in TCFEY (tris-citric acid-fructose 20% egg yolk), and two aliquots were prepared at a final concentration of 100 × 10 6 spz/ml, one maintained at room temperature (22 °C) and the other at 5 °C. In the first experiment, the toxicity of sucrose diluents on the sperm was analysed; sperm samples at different temperatures were diluted (1:2) in TCF-BSA 2% (control) or in the same extender supplemented with various sucrose concentrations (0.4 M, 0.6 M and 0.8 M). The effects of vitrification were studied in the second experiment, where sperm samples were mixed with different concentrations of cryoprotectants (sucrose) and vitrified by being plunged directly into liquid nitrogen. In both experiments, the sperm quality was assessed by measuring motility, morphology, membrane functionality (HOST), viability, acrosome integrity and DNA fragmentation. The toxicity test revealed significant differences (p ≤ 0.05) when different sucrose concentrations were used; lower total and progressive motility, normal morphology and membrane functionality were noted when sucrose concentration was higher, compared to the control treatment. Samples maintained at room temperature showed significantly (p ≤ 0.05) higher viability than samples stored at 5 °C. In contrast, although the quality of vitrified sperm was drastically decreased in comparison with fresh sperm, sucrose was associated with greater total motility, viability and membrane functionality. This improvement was closely linked to the temperature at which the sperm had been previously maintained, showing higher values when sperm was stored at 5 °C. The main conclusions to be drawn from the study are therefore that sucrose shows promising potential as a cryoprotectant, and storing samples at 5 °C is linked to improved sperm quality following vitrification. [ABSTRACT FROM AUTHOR]

This is the first report to examine the effect of iodixanol (OptiPrep TM ) on cryosurvival of buffalo spermatozoa. A total of thirty ejaculates (five ejaculates from each bull) from six buffalo bulls were used for this experiment. Each ejaculate was divided into four aliquots and diluted in freezing extender supplemented with different concentrations of OptiPrep TM (0, 1.25, 2.5 and 5%) and then cryopreserved. The semen quality variables were evaluated before and after freezing of the semen. There were no effects of OptiPrep TM ( P > 0.05) on sperm kinetics, motility, abnormality and membrane integrity of fresh extended spermatozoa. However, after freeze-thaw, sperm motility, plasma membrane integrity and distance travelled in cervical mucus of 2.5% OptiPrep TM treated samples showed significantly higher ( P < 0.05) compared to other treated and control samples. No significant differences ( P > 0.05) were seen in sperm abnormality and acrosomal integrity of treated and control frozen-thawed samples. The total antioxidant capacity of 2.5 and 5% OptiPrep TM treated frozen-thawed sperm were found to be higher ( P < 0.05) as compared to other groups; whereas the MDA level in OptiPrep TM treated sperm was significantly lower than the control ( P < 0.05). In incubation test, 2.5% OptiPrep TM proved to be better in preservation of sperm motility as compared to other treated and control samples. In conclusion, the present study has shown that iodixanol has the ability protect spermatozoa against oxidative stress and resulting overall improvement in the post-thaw semen quality. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation often leads to physical cell damage through ice crystal formation. This study evaluates the improvements to freezing extender cryoprotective activity due to antifreeze protein (AFP) addition, which primarily acts on ice crystal formation, through investigating the post-thaw sperm properties of Okinawan native Agu pig. Six individual boar sperm samples were diluted with the freezing extender supplemented with 1 μg/mL of AFP I or AFP III and then subjected to cryopreservation. Treatment with AFP I during the freezing procedure had no improvement for any characteristics after thawing compared to untreated sperm. In contrast, the addition of AFP III to the freezing extender strongly increased sperm motility, mitochondria and cell membrane integrity, and the acrosomal proteolytic activity of frozen-thawed sperm in 5 of 6 individuals (P < 0.05). Furthermore, cryoinjury prevention by AFP III significantly enhanced sperm viability (by ATP content), and maintained DNA quality and in vitro sperm penetrability compared with AFP I treatment (P < 0.05). These findings demonstrate that AFP III addition to the freezing extender of boar sperm is more effective in maintaining sperm characteristics than the extender without AFP III or AFP I, despite individual differences in response. [ABSTRACT FROM AUTHOR]

In the study, melatonin, a known antioxidant pineal peptide was used as an additive in the tris-egg yolk glycerol-based semen extender in Hariana bull semen and post-thaw sperm characters were evaluated. In the study, Group I was a control without melatonin; and Group II, III, and IV were having 0.5 mM, 1 mM, and 2 mM melatonin/80 × 106 spermatozoa, respectively were treatment groups. Thirty-two semen ejaculates from 4 Hariana bulls were processed for freezing and post-thaw sperm characteristics were evaluated. Sperm motility, velocity, the viability with intact membrane, and total antioxidant capacity were markedly (P < 0.05) improved in Group IV compared to all other groups. The lipid peroxidation and total protein carbonylation were substantially (P < 0.05) decreased in Group IV compared to all other groups. The population of cryocapacitated, acrosome-reacted, and apoptotic-like spermatozoa were significantly (P < 0.05) decreased in Group IV. Further, the relative band intensity of 74 kDa protein and percent of spermatozoa showing positive immune reactivity to tyrosine-phosphorylated proteins was decreased in Group IV. The progesterone-receptor ligand binding, in vitro capacitation response, and Vanguard distance were markedly (P < 0.05) improved in Group IV. In summary- Group IV having 2 mM melatonin was found to be optimal in providing cryoprotective effects to Hariana bull spermatozoa after freezing-thawing and can be suitably used as a semen additive during semen cryopreservation. • Melatonin in semen extender lowers oxidative damage to spermatozoa. • Melatonin in semen extender lowers cryocapacitation-like changes in spermatozoa. • Melatonin ion semen extender minimizes apoptotic-like changes in spermatozoa. • 2 mM melatonin can be used as an additive in Tris-egg yolk based semen extender. [ABSTRACT FROM AUTHOR]

The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1–1.5 yr, n = 6) and post-pubertal (3–7 yr, n = 5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200 mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell population, and viable population were assessed after 1 and 3 months of cryopreservation. The viable UTF1-positive population of pre-pubertal stallion germ cells was also measured using immunocytochemistry after 1 and 3 months of cryopreservation. As expected, the viability, cell population, and viable cell population were significantly reduced after 1 and 3 months of cryopreservation. At the pre-pubertal stage, the addition of trehalose or PEG to 10% DMSO did not show any effect on the viability, cell population, viable cell population, or viable UTF1-positive germ cells at either 1 or 3 months after cryopreservation. However, at the post-pubertal stage, the viable population was significantly higher in germ cells that were cryopreserved with 5% or 10% PEG, than in the cells cryopreserved with 10% DMSO only. In conclusion, PEG at 5% or 10% added to 10% DMSO serves as an optimal cryoprotectant agent for the cryopreservation of germ cells from post-pubertal stallions. [ABSTRACT FROM AUTHOR]

This study evaluates freezing protocol with suitable cryoprotectants and their effects on the fertility potential of sperm in the cryopreserved spermatophores of Macrobrachium rosenbergii . Spermatophores, collected using electroejaculation, were suspended in dimethyl sulfoxide (DMSO), propylene glycol (PG), methanol, glycerol and ethylene glycol (EG) at different concentrations (10, 15 & 20% v/v), prepared in sterile-filtered pond water. Based on the cryoprotectant toxicity assay, DMSO and PG were used individually as well as in combination with three freezing protocols (i.e. −1.5, −3 and −5 °C/min and to final temperature of −39 °C) and plunged into liquid nitrogen at −196 °C. After 90 days of storage (−196 °C) thawing was done at 35 °C in a water bath for 1 min. Results showed that fresh and cryopreserved spermatophores held for 90 days registered sperm viability of 91.4 ± 2.9% and 50.4 ± 1.9% respectively. Further, fertility potential of sperm was assessed based on acrosome reactivity using calcium ionophore (A23187). Observations indicated that cryopreserved sperm registered 28.3 ± 2.2% of acrosome reactivity compared to freshly collected spermatophores (85.3 ± 2.5%). Thus, one-step slow cooling rate of −1.5 °C/min between 27 °C and −39 °C stored in liquid nitrogen at −196 °C with DMSO (10%) + PG (10%) seems to be amenable for cryopreservation of spermatophores, compared to other cooling rates. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation facilitates storage and transport for use in artificial reproduction technologies. Cryopreservation processing, however, exposes cells to stress resulting in cellular damage compromising sperm function. Cryoprotective agents are needed to minimize cryopreservation injury, but at higher concentration they are toxic to cells. In this review, we describe cryoinjury mechanisms, and modes of action of different types of cryoprotective agents. Furthermore, measures are discussed how to minimize toxic effects caused by adding and removing cryoprotective agents. Cryoprotective agents can be divided into permeating and non-permeating agents. Permeating agents such as glycerol can move across cellular membranes and modulate the rate and extent of cellular dehydration during freezing-induced membrane phase transitions. Permeating protectants provide intracellular protection because they are preferentially excluded from the surface of biomolecules thereby stabilizing the native state. Non-permeating agents can be divided into osmotically active smaller molecules and osmotically inactive macromolecules. Both, permeating and non-permeating protectants form a protective glassy state during freezing preserving biomolecular and cellular structures. Freezing extenders for sperm contain salts, buffer compounds, sugars, proteins and lipids, and typically contain glycerol as the main permeating cryoprotective agent providing intracellular protection. Non-permeating protectants including sugars and proteins are used as bulking agents and to increase the glass transition temperature of the freezing extender. Ultra-heat-treated milk and egg yolk are frequently added as membrane modifying agents to enhance the inherent sperm cryostability. The protocol how to use and add cryoprotectants is a compromise between their beneficial and potentially detrimental effects. [ABSTRACT FROM AUTHOR]

The current study determined the effect of the egg-yolk (phospholipid source) level (egg yolk [20% EY] vs. skim-milk + egg yolk [SM + 2% EY]), cryoprotectant (glycerol [Gly] vs. glycerol + methylformamide [Gly + MF]), and pre-freeze cooling rate (–0.1 vs. –1 vs. −5 °C/min) on post-thaw stallion sperm quality. In Experiment 1, ejaculates (n = 27) from 9 stallions (3 ejaculates each) with varied sperm quality (High, Average, or Low) were frozen in EY-Gly, SMEY-Gly, EY-Gly + MF, or SMEY-Gly + MF extenders. Sperm in each group were cooled from 22° to 5°C using either −0.1 °C/min or −1 °C/min linear cooling rates prior to freezing. In Experiment 2, ejaculates (n = 24) from 12 stallions (2 ejaculates each) with High or Average sperm quality were frozen in EY-Gly, EY-Gly + MF, or in BotuCrio (BC) extenders. Sperm in each group were cooled from 22° to 5°C using either –1 or −5 °C/min linear cooling rates prior to freezing. In Experiment 1, for stallions with High or Average sperm quality, either cooling rate generally resulted in lower sperm quality for the SMEY-based extenders than for the EY-based extenders (P < 0.05). Stallions with Low sperm quality were unaffected by any experimental treatment (P > 0.05). In Experiment 2, a −5 °C/min cooling rate yielded lower sperm quality in BC than in EY-Gly or EY-Gly + MF groups (P < 0.05); however, a −1 °C/min cooling rate yielded similar sperm quality among these treatments (P > 0.05). In summary, the phospholipid level in the freezing extender and the pre-freeze cooling rate, but not the penetrating cryoprotectant, affected the post-thaw quality of stallion sperm. • 20% egg yolk (EY) yielded higher post-thaw stallion sperm quality than 2% EY. • Glycerol or glycerol and methylformamide yielded similar post-thaw sperm quality. • Pre-freeze cooling rate influenced post-thaw sperm quality in 2% EY-extenders. • Pre-freeze cooling rate did not affect post-thaw sperm quality in 20% EY-extenders. • Pre-freeze cooling rate was not affected by penetrating cryoprotectants. [ABSTRACT FROM AUTHOR]

Czech Golden Spotted Hen (CZH) is the only original national Czech hen breed, classified among genetic resources and protected under a national rescue program. Sperm cryopreservation procedures for this breed are essential to create a sufficient genetic reservoir, as there were less than 260 CZH birds (both males and females) in this program in 2020. The aim of the study was to compare commercial fresh semen extenders supplemented with a defined cryoprotective agent as to suggest important tool for cryopreservation procedure of original Czech hen breed. Ejaculates were obtained from four adult roosters (Gallus gallus domesticus) collected by dorso-abdominal massage technique twice a week. Semen samples meeting initial standardss were mixed together and diluted in commercially available extenders with cryoprotective agent: Poultry media®, Raptac®, and NeXcell® supplemented with 9% N-Methylacetamide. Diluted semen was frozen in 0.25 ml plastic straws exposed to liquid nitrogen vapours (5 cm above) for 10 min and subsequently plunged into liquid nitrogen for storage. Sperm samples were thawed in a water bath tempered at 5°C for 100 s. Sperm parameters of viability (VIA; expressed by the plasma membrane integrity, and acrosome status) and sperm motility (MOT), were assessed using flow cytometry and mobile computer-assisted sperm analyzer – mCASA (iSperm®) before and after cryopreservation. Poultry media® (VIA =34.80%, MOT =51.95%) and Raptac® (VIA =38.33%, MOT = 51.25%) with cryoprotective supplement had comparable results with highest cryoprotective efficiency. In contrast, significantly lower VIA and MOT parameters were detected for CZH roster spermatozoa froze in NeXcell® extender compared to Poutry media® or Raptac®. Our study identified appropriate commercial fresh semen extenders modified for cryopreservation procedure of sperm from the original Czech hen breed. It will be, however, necessary to verify our promising in vitro results with artificial insemination in future. [ABSTRACT FROM AUTHOR]

Using cryopreservation techniques can increase the effectiveness of reproducing cultured fish species by ensuring a dependable supply of sperm, although the quality of the sperm could be impacted by the procedures involved. The goal of this study was to investigate the effect of purified seminal plasma transferrin (Tf), bovine serum albumin (BSA), and antifreeze protein (AFP) types I and III at 1 µg mL -1 on relevant characteristics of cryopreserved sperm from common carp Cyprinus carpio. We compared oxidative stress indices, antioxidant activity, and DNA fragmentation of fresh sperm to that frozen with extender only or with Tf, BSA, or AFP types I and III. Fresh sperm had significantly lower levels of thiobarbituric acid reactive substances (TBARS) compared to samples that underwent cryopreservation without protein treatment, which resulted in 0.54 ± 0.06 nmol/10 8 cells of TBARS. Carbonyl derivatives of proteins (CP) decreased significantly (ANOVA; P > 0.05) in carp sperm with addition of Tf, AFPI, and AFPIII. Significant differences in superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) activity were seen in sperm supplemented with Tf, BSA, AFPI, and AFPIII from those without. Significantly less DNA damage, expressed as percent tail DNA (11.56 ± 1.34) and olive tail moment (0.59 ± 0.13), was recorded in samples cryopreserved with Tf. The findings indicated that addition of Tf, BSA, AFPI, or AFPIII to cryopreservation medium is beneficial to sperm preservation. The mechanisms through which these proteins act positively on sperm need to be further investigated., Competing Interests: Conflict of interest statement The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The present study investigates the effects of five cryoprotectants (CPAs) and cryoprotectant combinations on the post-thaw total motility, progressive motility, viability, mitochondrial membrane potential and acrosome integrity in stallion spermatozoa. In Experiment 1, the objective was to compare the impact of different concentrations (2.5%, 3.5% and 5%) of a single CPA, including glycerol (Gly), ethylene glycol (EG), dimethyl sulphoxide (DMSO), methyl formamide (MF), and dimethylformamide (DMF) for stallion spermatozoa cryopreservation. In Experiment 2, two or more CPAs were used to assess whether this improved post-thaw spermatozoa quality. Gly, MF and DMF, were used to prepare seven combinations of freezing extender with different mixtures of cryoprotectant, and the 3.5% Gly, MF and DMF were used as a control group. The results show that post-thaw total motility, progressive motility, viability, and mitochondrial membrane potential for all concentrations of EG and DMSO were less than the 3.5% and 5% Gly and MF and 2.5% and 3.5% DMF ( P < 0.05). Use of the 3.5% concentration resulted in the greater post-thaw total motility and progressive motility than the 2.5% and 5% concentrations for all CPAs. The results for the use of different combinations of cryoprotectant indicate there are differences in progressive motility and viability. The viability with the use of Gly(2/3) + MF(1/3) was 44.65% and was greater than the Gly(1/3) + MF(1/3) + DMF(1/3) (30.96%), MF(2/3) + DMF(1/3) (35.05%), Gly (32.21%) and MF(33.76%) ( P < 0.05). The progressive motility with the use of the MF(2/3) + Gly(1/3) combination was 36.0% and was greater than in the DMF (25.0%) and MF(2/3) + DMF(1/3) (22.7%) ( P < 0.05). These results suggest that using the appropriate cryoprotectant combination instead of a single cryoprotectant can improve horse spermatozoa cryopreservation. [ABSTRACT FROM AUTHOR]

This study examined the effect of cryoprotectants on DNA integrity, antioxidant defense, and resistance to oxidative stress in cryopreserved sterlet Acipenser ruthenus sperm. The freeze-thaw process significantly influenced sperm motility, with significant differences among cryoprotectants. In vitro exposure of cryopreserved sperm to the xanthine–xanthine oxidase (X–XO) system as a model reactive oxygen species inducer resulted in a lesser motility rate and velocity compared to the control, and there was a decrease in these variables in a time- and dose-dependent manner. The greatest X (0.6 mM)–XO (0.05 U/mL) concentration and incubation period (30 min) was associated with 62% DNA fragmentation in sperm cryopreserved with 10% ethylene glycol (EG). The maximum lipid peroxidation (LPO) and carbonyl derivatives of proteins (CP) was also observed in sperm cryopreserved with 10% EG and exposed to the X–XO system at a concentration of 0.6 mM X–0.05 U/mL XO. The frozen/thawed sperm containing 10% EG and that with 10% dimethyl sulfoxide (DMSO) had a significant enhancement of superoxide dismutase (SOD) and glutathione reductase (GR) activity. The current study confirms that EG is not effective for cryopreservation, and sterlet sperm were highly sensitive to free radicals after cryopreservation with EG. [ABSTRACT FROM AUTHOR]

This study was designed to identify the most effective non-permeable cryoprotectant (CPA) for the cryopreservation of rabbit semen by comparing the effects of different concentrations of low-density lipoproteins (LDL) on post-thaw sperm quality with those of whole egg yolk or sucrose. In a second experiment, the performance of the non-permeable CPAs identified as most effective was assessed in vivo by determining reproductive performances. Pooled semen samples were diluted to a ratio of 1:1 (v:v) in freezing extender (Tris-citrate-glucose and 16% dimethylsulfoxide as permeable CPA) containing as non-permeable CPAs 6, 8, 10 or 15% LDL from egg yolk, 0.1 M sucrose, or 15% egg yolk. The semen was loaded in 0.25 mL straws and frozen in liquid nitrogen vapor. After thawing, we determined sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Our results clearly revealed a significant effect of LDL concentration on semen quality. Also, at an optimal concentration of 10%, motility and acrosome integrity were improved over the values recorded for egg yolk ( P < 0.05). Based on the in vitro data, 3 groups of does ( n = 30 each) were inseminated with fresh semen or semen frozen using sucrose or 10% LDL. Sucrose led to a significantly higher conception rate than LDL and reproductive performance was similar to that observed for fresh semen. Our findings indicate the markedly better performance of sucrose in vivo as a non-permeable CPA for the cryopreservation of rabbit semen. [ABSTRACT FROM AUTHOR]

The objectives of this study were to compare glycerol (G) and ethylene glycol (EG) at different concentrations and trehalose (T) or cysteine (C; with/without) in Tris extender for cryopreservation of bull semen. Twenty-four ejaculates obtained from three bulls were included in the study. Each ejaculate was divided into four equal aliquots and diluted using both of the Tris extenders with G (5% or 7%) or EG (3% or 5%). After that, each extenders were divided into three equal aliquots and diluted using both of the 5 mM C or 25 mM T, and control (without additives) was cooled to 4 °C and frozen in 0.25 ml French straws. The addition of 3% and 5% EG without antioxidants resulted in the least Computer-Assisted Sperm motility Analysis (CASA) motility as compared with the other groups. Treatment with 25 mM T in 3% EG beneficially effected acrosome morphology as compared with the other groups. Also, treatment with 3% EG with 25 mM T and 5% EG resulted in a greater rate of total abnormalities. Treatment with 3% G yielded a slightly greater percentage of membrane integrity by Hypo-Osmotic Swelling Test (HOST) assessment than that of the other groups. Treatment with 3% EG with 5 mM C resulted in the greatest concentration of malondialdehyde (MDA). The glutathione peroxidase (GPx) antioxidant activity was increased in the C-treatment groups when compared to the other groups. Treatment with 5% EG and 5 mM C resulted in less chromatin damage and detrimental impacts on tail moment. Treatment with 5% EG led to greater non-return rates of inseminated cows. However, this result was not considered to be statistically important. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effect of different cryo-protectants (glycerol, dimethylacetamide and dimethylformamide alone or combined and added to lactose–egg yolk extender) on the viability of frozen/thawed semen from the Piau breed as assessed by in vitro testing. Frozen semen samples (n =20) were used from five male swine. Five different freezing extenders, including 2% glycerol (Group 1 – G), 2% glycerol and 3% dimethylacetamide (Group 2 – GA), 2% glycerol and 3% dimethylformamide (Group 3 – GF), 5% dimethylacetamide (Group 4 – A) and 5% dimethylformamide (group 5 – F), were evaluated. To assess post-thawing sperm quality, sperm motility and morphology were evaluated. Sperm viability was determined using the hypoosmotic swelling test, supravital staining, and a fluorescent assay (carboxyfluorescein diacetate and propidium iodide). The mean total sperm motility of semen immediately after thawing was 46.2±1.3, 57.7±1.5, 53.2±2.1, 51.7±1.2, and 46.5±1.6% for groups 1–5, respectively. Groups 2 (GA) and 3 (GF) had greater motility values (P <0.05). Fluorescent assay values of 22.3±2.3%, 35.2±3.7%, 30.8±3.4%, 36.6±3.7%, and 26.5±3.8% were obtained for Groups 1–5, respectively, showing that Group 4 (A) sperm had greater viability than those from Group 1 (G), although there was no differences between the other treatments (P >0.05). The other complementary tests (hypoosmotic swelling test and supra-vital staining) demonstrated that sperm in Groups 2 (GA), 3 (GF) and 4 (A) had the greatest viability and there were no significant differences among these three groups (P >0.05). The most effective cryo-protectant combinations likely minimized and controlled the deleterious processes that occur in the sperm cell during freezing/thawing, thus improving post-thawing sperm viability. In conclusion, the combination of amides (3%) and glycerol (2%) or dimethylacetamide (5%) alone were more efficient at cryo-protection than glycerol alone for semen freezing in the Piau swine breed. [ABSTRACT FROM AUTHOR]