Sperm quality and the morphology of cryopreserved testicular tissues recovered post-mortem from diverse wild species

This study compared the effects of slow and fast freezing of testicular tissue of wild animals collected at post-mortem on testicular structure and testicular sperm. The testes of seven animals that had died in captivity; three felids (jungle cat, lion and leopard), two cervids (rusa deer and fea’s muntjac) and two bovids (Sumatran serows) were cryopreserved using slow- and fast-freezing protocols. There were greater reductions in the integrity of the sperm membrane and DNA in tissues cryopreserved using slow freezing compared to fast freezing (membrane integrity reduced by 21.5 ± 12.4% vs. 13.0 ± 6.9%, P = 0.11 and DNA integrity reduced by 22.7 ± 16.3% vs. 6.6 ± 6.3%, P = 0.13). Histologically, there were similar degrees of detachment and shrinkage of the seminiferous tubules whereas, TUNEL assay revealed a tendency towards more apoptotic changes in the intra-tubular cells of tissues frozen using fast freezing compared to slow freezing (P = 0.09). In conclusion, fast freezing tended to cause less damage to testicular sperm but its protective effect on intra-tubular cells was likely compromised. This is the first report of gamete recovery in the wild and of the comparison in various wildlife species, between testicular tissues cryopreserved using different protocols.     

海藻酸微囊替代DMSO和FBS的STO细胞冷冻保存研穷

目的：改进现有的细胞冷冻保存方法,建立一个不舍二甲基亚砜（DMSO）和血清（FBS）的高效冷冻保存方法,为细胞治疗等临床实践提供优质细胞。方法：海藻酸微囊包埋鼠胚成纤维细胞（STO细胞）后用不含DMSO和FBS的冷冻保存液进行冷冻保存。,设四个对照组：添加10％DMSO和20％FBS的组、仅添加10％DMSO的组、仅添加20％FBS、DMSO和FBS均不添加组。在冷冻前后对各实验组细胞用台盼兰染色,进行细胞计数,计算细胞存活率,同时利用溴乙锭的二聚物（EthD）、钙黄绿素-AM（Calcein—AM）进行染色观察细胞的形态,且进一步验证细胞存活率;解冻复苏后用MTT法评估细胞的增殖速度和生长活力。结果：冷冻保存30天后对各组的细胞数量、细胞存活率、细胞形态和解冻复苏后细胞的生长活力进行比较发现,海藻酸微囊包埋冷冻组的细胞数、细胞存活率、细胞形态和生长活力均与添加DMSO和FBS的组之间无显著性差异,而与其它三个对照组呈显著性差异。结论：使用海藻酸微囊替代DMSO和FBS保存STO细胞,能有效的维持细胞形态、数量、存活率,同时不影响细胞的生长活力,从而建立了一个不含DMS0和FBS的高效冷冻保存方法。     

Successful sperm cryopreservation of the brown-marbled grouper,Epinephelus fuscoguttatus using propylene glycol as cryoprotectant

This study developed the cryopreservation of brown-marbled grouper spermatozoa for practical application. We examined 32 cryodiluents, developed from four types of cryoprotectants [propylene glycol (PG), dimethyl-sulphoxide (Me₂SO), dimethyl-acetamide (DMA) and ethylene glycol (EG)] at four concentrations of 5, 10, 15 and 20% in combination with two extenders [Fetal bovine serum (FBS) and artificial seminal plasma (ASP). Cooling rates were examined by adjusting the height of straws (2.5–12.5 cm) from the liquid nitrogen (LN) vapor and cooled for 5 min before immersion into LN. DNA laddering was used to detect DNA damage in cryopreserved sperm. In fertilization trials, 0.5 g of eggs was mixed with cryopreserved sperm stored for 30 days in LN. The best motility of post-thaw sperm was achieved using 15% PG + 85% FBS (76.7 ± 8.8%); 10% PG + 90% FBS was also effective as cryodiluent. Generally, FBS gave better post-thaw motility compared to ASP. The optimum cooling rate was at 17.6 °C min⁻¹ obtained by freezing at the height of 7.5 cm surface of LN. The results obtained showed that cryopreserved sperm of brown-marbled grouper suffered slight DNA fragmentation, which resulted in significantly lower motility. However, the fertilization (90.9 ± 0.5%), hatching (64.5 ± 4.1%) and deformity rates (3.8 ± 0.2%) obtained from cryopreserved sperm showed no significant difference with fresh sperm. These findings show that the developed protocol for cryopreservation of brown-marbled grouper sperm was viable and will be useful for successful breeding and seed production of brown-marbled grouper.     

Inverse relationship between testicular proliferation and apoptosis in mammalian seasonal breeders

Seasonal cycles of testicular activity occur in many mammals and can include transitions between total arrest and recrudescence of spermatogenesis. We hypothesize that involution and reactivation of testis result from two antagonistic processes, proliferation and programmed cell death (apoptosis), which are activated at different times. To test this hypothesis, quantitative measurements of both proliferation-specific marker and apoptotic produced nucleosomes have been compared with sperm and testosterone production in testes from adult roe deer during breeding and non-breeding seasons (May to September). Testes of brown hare were included from periods of testes regression (June to August) and recrudescence (November to December). The highest testicular weights in roe deer were found in the rutting period from late July to early August (27.25 +/- 8.56 g), corresponding with the highest number of testicular sperm/g parenchyma. The peak of sperm production coincided with a peak in testosterone concentration (1.19 +/- 0.53 microg/g testis). The maximum level of proliferation-specific marker was also found during the breeding season (98.6 +/- 58.2 U/g testis in comparison to 20.1 +/- 22.0 U/g in the prerutting period). In contrast, the most significant apoptosis was observed in the nonbreeding season than the breeding period (71.11 +/- 5.79 U/mg testis and 18.88 +/- 6.79 U/mg, respectively). Testicular proliferation was low in the brown hare (0.061 +/- 0.062 U/g) during involution of the testes. It was newly activated in November and December (0.85 +/- 0.33 U/g), preceding the increase in testicular volume. Testosterone production increased in conjunction with testicular proliferation. At this time, testicular apoptosis was significantly lower (14.16 +/- 2.12 U/mg testis) than during the period of pronounced testicular regression (30.16 +/- 19.95 U/g). These results suggest that regulation of seasonal testicular activity is characterized by an inverse relationship of proliferation and apoptosis.     

Neuropeptides to replace serum in cryopreservation of mesenchymal stromal cells?

Background aimsThe therapeutic potential of human mesenchymal stromal cells (MSCs) has generated considerable interest in a wide variety of areas. MSC banking is feasible, but the optimal technique of cryopreservation remains to be determined.MethodsTo reduce dimethyl sulfoxide (DMSO) concentration in cryopreservation medium, DMSO was replaced with sucrose or trehalose. To increase cell survival and proliferation rates after thawing and to eliminate the need for fetal bovine serum (FBS), neuropeptides of the vasoactive intestinal peptide/glucose-dependent insulinotropic peptide/pituitary adenylate cyclase activating polypeptide family were added to the cryopreservation medium. Cell survival was analyzed by a trypan blue dye exclusion assay. Cell proliferation of cryopreserved MSCs was determined after 7 days of culture.ResultsNo significant differences in cell survival rates were detected between cryopreservation solutions with 5% and 10% DMSO, independently of the addition of trehalose or sucrose. Cell proliferation rates tended to be highest when MSCs were frozen in 5% DMSO + trehalose. FBS could be replaced by human albumin (HA) without loss in cell survival and proliferation potential. With FBS, the addition of neuropeptides could increase cell survival and proliferation rates. Without FBS or HA, cell survival and proliferation rates in the presence of neuropeptides were comparable to rates achieved with FBS or HA.ConclusionsClassic cryopreservation with 10% DMSO could be replaced by 5% DMSO + 30 mmol/L trehalose. FBS could be replaced by HA or neuropeptides without loss in cell survival and proliferation potential. The addition of neuropeptides in the cryopreservation medium containing FBS could increase the cell proliferation rate and consequently cellular output.     

The effects of extenders and sperm‐egg ratios on fertilizing ability of cryopreserved testicular sperm of northern pike (Esox lucius L.)

The effects of four different extenders and two sperm‐to‐egg ratios on fertilizing ability of cryopreserved testicular sperm of northern pike (Esox lucius L.) were tested in the present study. Testicular sperm was diluted with each of the four extenders (0.6 m sucrose + 15% DMSO, 0.3 m glucose + 15% DMSO, 0.6 m sucrose + 10% methanol and 0.3 m glucose + 10% methanol, all supplemented with 10% egg yolk and 1.7 g KCl L^?1) and frozen in 0.5‐ml straws at 2 cm above the surface of liquid nitrogen and thawed at 25°C for 30 s. Then 125 μl or 50 μl of frozen‐thawed testicular sperm was poured onto about 1250 eggs for fertilization. The results showed that both sperm‐to‐egg ratio and diluent had no significant influence on cryopreservation efficiency of testicular sperm, whereas cryoprotectant had a significant influence. The highest fertilization rate (92.2%) was obtained from testicular sperm cryopreserved in glucose‐based extender containing 10% methanol at a sperm‐to‐egg ratio of 1 × 10⁶ spermatozoa per egg. The results indicated that glucose‐based extender containing 10% methanol, 10% egg yolk and 1.7 g KCl L^?1 was a useful combination.     

Optimization of cryoprotectants for cryopreservation of rat hepatocyte

Rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing either fetal bovine serum (FBS), glycerol, dimethyl sulfoxide (DMSO), sucrose or a mixture of these as a cryoprotectant. The best survival was with 10% (v/v) DMSO containing 30% (v/v) FBS using 5 x 10(5) hepatocytes ml(-1) at -70 degrees C for 5 d on type I collagen-coated dishes. After thawing, the cell viability was 81% determined by the MTT-test. The cryopreserved hepatocytes had the capacity of albumin synthesis similar to hepatocytes without cryopreservation. This result shows that cryopreservation of rat hepatocyte can be used for the evaluation of hepatic functions.     

Cryopreservation of immature porcine testis tissue to maintain its developmental potential after xenografting into recipient mice

The purpose of this study was to develop effective strategies for cooling and cryopreservation of immature porcine testis tissue that maintain its developmental potential. Testes from 1-wk-old piglets (Sus domestica) were subjected to 1 of 12 cooling/cryopreservation protocols: as intact testes, cooling at 4 °C for 24, 48, or 72 h (Experiment 1); as fragments, programmed slow-freezing with dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol (Experiment 2); or solid-surface vitrification using DMSO, glycerol, or ethylene glycol, each using 5-, 15-, or 30-min cryoprotectant exposure times (Experiment 3). For testis tissue xenografting, four immunodeficient recipient mice were assigned to each protocol, and each mouse received eight grafts. Recipient mice were killed 16 wk after grafting to assess the status of graft development. Based on morphology and in vitro assessment of cell viability, cooling of testis tissue for up to 72 h maintained structural integrity, cell viability, in vivo growth, and developmental potential up to complete spermatogenesis comparable with that of fresh tissue (control). In frozen-thawed testis tissues, higher numbers of viable cells were present after programmed slow-freezing using glycerol compared with that after DMSO or ethylene glycol (P < 0.001). Among the vitrified groups, exposure to DMSO for 5 min yielded numerically higher viable cell numbers than that of other groups. Cryopreserved tissue fragments recovered after xenografting had normal spermatogenesis; germ cells advanced to round and elongated spermatids after programmed slow-freezing using glycerol, as well as after vitrification using glycerol with 5- or 15-min exposures, or using DMSO for a 5-min exposure.     

Cryopreservation of calf testicular tissues with knockout serum replacement

To enrich bovine gonocytes from cryopreserved testicular tissues, the cryoprotection effects of the freezing media containing knockout serum replacement (KSR) were examined. Using Minimum essential medium (MEM) + 10% dimethyl sulfoxide (Me₂SO) as the basic medium, calf testicular tissues were cryopreserved in media containing 0, 5, 10, 20, 40, 90% KSR and 5% fetal bovine serum (FBS) respectively. Morphologically, the seminiferous cords and interstitium were well preserved in all groups. The gonocytes were all glial cell line-derived neurotrophic factor (GDNF) family receptor α-1 (GFRα-1) positive. The recovery rates in all KSR groups were higher than that of the 10% Me₂SO group, while comparable to the 5% FBS group. The enriched gonocytes expressed gonocyte marker GFRα-1 typically. Collectively, supplementation of 5–10% KSR can achieve comparable cryoprotective effects with using 5% FBS, which is useful in future study due to its defined formulation that is more consistent in quality and stable in supply.     

Comparison of cryosurvival and spermatogenesis efficiency of cryopreserved neonatal mouse testicular tissue between three vitrification protocols and controlled-rate freezing

Grafting of cryopreserved testicular tissue is a promising tool for fertility and testicular function preservation in endangered species, mutant animals, or cancer patients for future use. In this study, we aimed to improve the whole neonatal mouse testicular tissue cryopreservation protocols by comparing cryosurvival, spermatogenesis, and androgen production of grafted testicular tissue after cryopreservation with three different vitrification protocols and an automated computed controlled-rate freezing. Whole neonatal mouse testes were vitrified with various vitrification solutions (V1) 40% EG + 18% Ficoll + 0.35 M Sucrose, (V2) DAP 213 (2 M DMSO + 1 M Acetamid + 3 M PG), or (V3) 15% EG + 15% PG + 0.5 M Sucrose (total solute concentration V1:74.34%, V2:44.0%, and V3:49.22% wt/vol). Alternatively, neonatal testicular tissue was also frozen in 0.7 M DMSO +5% fetal bovine serum using controlled-rate freezing and compared to fresh grafted testicular tissue, sham grafted controls, and the vitrification protocol groups. Fresh (n = 4) and frozen-thawed (n = 4) testes tissues were grafted onto the flank of castrated male NCr Nude recipient mouse. The grafts were harvested after three months. Fresh or frozen-thawed grafts with controlled-rate freezing had the highest rate of tissue survival compared to other vitrified protocols after harvesting (p < 0.05). Both controlled-rate freezing and V1 protocol groups displayed the most advanced stages of spermatogenesis with elongated spermatids and spermatozoa in 17.6 ± 1.3% and 16.3 ± 1.9% of seminiferous tubules based on histopathological evaluation, respectively. Hosts of the testicular graft from controlled-rate freezing had higher levels of serum testosterone compared to all other vitrified-thawed graft groups (p < 0.05). This study shows that completed spermatogenesis from whole neonatal mouse testes were obtained when frozen with controlled-rate freezing and V1 vitrification solution and that testicular cryopreservation efficacy vary with the protocol and vitrification technique.     

Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific Oysters, Crassostrea gigas

Despite some 26 published reports addressing oyster sperm cryopreservation, systematic factor optimization is lacking, and sperm cryopreservation has not yet found application in aquaculture on a commercial scale. In this study, the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization of shipped sperm samples from diploid oysters. Evaluation of cooling rates revealed an optimal rate of 5 degrees C/min to -30 degrees C followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations suggested that a low concentration (2%) of polyethylene glycol (FW 200) was effective in retaining post-thaw motility and fertilizing capability when combined with permeating cryoprotetcants such as dimethyl sulfoxide (DMSO), methanol (MeOH), and propylene glycol (P-glycol). However, polyethylene glycol alone was not as effective as MeOH, DMSO, and P-glycol when using the same methods. The highest post-thaw motility (70%) and percent fertilization (98%) were obtained for samples cryopreserved with 6% MeOH. However, this does not exclude other cryoprotectants such as DMSO or P-glycol identified as effective agents in other studies. There was no significant difference in post-thaw motility between straw sizes of 0.25- and 0.5-ml. Equilibration time (exposure to cryoprotectant) of 60 min could be beneficial when the cryoprotectant concentration is low and solution is added in a step-wise fashion at low temperature. Differences in post-thaw sperm quality (e.g., motility or percent fertilization) among individual males were evident in this research. As a consequence, a generalized classification describing males with different tolerances (broad, intermediate, and narrow) to cryopreservation was developed. This classification could be applied to strain or species differences in tolerances to the cryopreservation process. The present study demonstrated that oyster sperm could be collected and shipped chilled to another facility for cryopreservation, and that it could be shipped back to the hatchery for fertilization performed at a production scale yielding live larvae with >90% fertilization. Given the existence of facilities for commercial-scale cryopreservation of dairy bull sperm, the methods developed in the present study for oysters provide a template for the potential commercialization of cryopreserved sperm in aquatic species.     

Effect of different cryoprotectant agents on spermatogenesis efficiency in cryopreserved and grafted neonatal mouse testicular tissue

Restoration of male fertility associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The purpose of this study was to test the effects of four different cryoprotectant agents (CPA) on spermatogenesis and steroidogenesis in cryopreserved and allotransplanted neonatal mouse testicular tissue. Hank’s balanced salt solution (HBSS) with 5% fetal bovine serum including either 0.7 M dimethyl sulfoxide (DMSO), 0.7 M propylene glycol (PrOH), 0.7 M ethylene glycol (EG), or glycerol was used as the cryoprotectant solution. Donor testes were collected and dissected from neonatal pups of CD-1 mice (one day old). Freezing and seeding of the testicular whole tissues was performed using an automated controlled-rate freezer. Four fresh (non-frozen) or frozen–thawed pieces of testes were subcutaneously grafted onto the hind flank of each castrated male NCr nude recipient mouse and harvested after 3 months. Fresh neonatal testes grafts recovered from transplant sites had the most advanced rate of spermatogenesis with elongated spermatid and spermatozoa in 46.6% of seminiferous tubules and had higher levels of serum testosterone compared to all other frozen–thawed-graft groups (p < 0.05). Fresh grafts and frozen–thawed grafts in the DMSO group had the highest rate of tissue survival compared to PrOH, EG, and glycerol after harvesting (p > 0.05). The most effective CPA for the freezing and thawing of neonatal mouse testes was DMSO in comparison with EG (p < 0.05) in both pre-grafted and post-grafted tissues based on histopathological evaluation. Likewise, the highest level of serum testosterone was obtained from the DMSO CPA group compared to all other cryoprotectants evaluated (p < 0.05). The typical damage observed in the frozen–thawed grafts included disruption of the interstitial stroma, intercellular connection ruptures, and detachment of spermatogonia from the basement membrane. These findings indicate that neonatal mouse testes were most effectively preserved when frozen with HBSS medium with DMSO and that the type of CPA is a significant factor to obtain the most advanced stages of spermatogenesis and steroidogenesis after cryopreservation, thawing, and transplantation of neonatal mouse testes.     

Effect of glycerol and dimethyl sulfoxide on cryopreservation of rhesus monkey (Macaca mulatta) sperm

Glycerol and dimethyl sulfoxide (DMSO) are widely used as penetrating cryoprotectants in the freezing of sperm, and various concentrations are applied in different species and laboratories. The present study aimed to examine the effect of these two cryoprotectants at different concentrations (2%, 5%, 10%, and 15% glycerol or DMSO) on rhesus monkey sperm cryopreservation. The results showed that the highest recovery of post-thaw sperm motility, and plasma membrane and acrosome integrity was achieved when the sperm was frozen with 5% glycerol. Spermatozoa cryopreserved with 15% DMSO showed the lowest post-thaw sperm motility, and spermatozoa cryopreserved with 15% glycerol and 15% DMSO showed the lowest plasma membrane integrity among the eight groups. The results achieved with 5% glycerol were significantly better for all parameters than those obtained with 5% DMSO. The functional cryosurvival of sperm frozen with 5% glycerol was further assessed by in vitro fertilization (IVF). Overall, 85.7% of the oocytes were successfully fertilized, and 51.4% and 5.7% of the resulting zygotes developed into morulae and blastocysts, respectively. The results indicate that the type and concentration of the penetrating cryoprotectant used can greatly affect the survival of rhesus monkey sperm after it is frozen and thawed. The suitable glycerol level for rhesus monkey sperm freezing is 5%, and DMSO is not suitable for rhesus monkey sperm cryopreservation.     

Sperm cryopreservation of the critically endangered olive barb (Sarpunti) Puntiussarana (Hamilton, 1822)

The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntiussarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsever’s solution prepared at 296 mOsmol kg⁻¹. Sperm were activated with distilled water (24 mOsmol kg⁻¹) to characterize motility. Maximum motility (90%) was observed within 15 s after activation, and sperm remained motile for 35 s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was ?287 mOsmol kg⁻¹. To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10 min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5 °C to −80 °C at 10 °C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7–15 days at −196 °C. The highest motility in thawed sperm 61 ± 8% (mean ± SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.     

Cell survival after cryopreservation of dissociated testicular cells from feline species

Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 μm and 20.5 ± 4 μm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.     

Proliferation and differentiation potential of cryopreserved human skin-derived precursors

Objectives: Skin‐derived precursors are recognized to be a potentially autologous and accessible source of neural precursor cells for drug screening or cell‐based treatments, in many neurological disorders. Thus, it is necessary to investigate appropriate methods for cryopreservation of such human skin‐derived precursors (hSKPs). The aim of this study was to evaluate different cryopreservation techniques for retention of hSKPs to discover an optimized protocol. Materials and methods: We cryopreserved hSKPs treated with 0%, 10%, 20%, 30% and 40% foetal bovine serum (FBS) and three concentrations of dimethylsulphoxide (DMSO) 5%, 10% and 15%, with two different storage periods in liquid nitrogen (2 days: short‐term storage; and 2 months: long‐term storage). Then, we assessed survival and proliferation levels of the cells after freeze–thaw processes, by viability measurement and colony‐forming assay. For detecting hSKPs, we used immunocytochemistry and RT‐PCR assessments. Results: Our findings indicated that hSKPs cryopreserved in 5% DMSO without FBS, had better survival and proliferation potentials compared to other working formulations. With various concentrations of cryoprotectants over different time periods, hSKPs retained their differentiation potentiality and were able to differentiate into neurons (NFM and βΙΙΙ tubulin‐positive), glial cells (GFAP‐positive) and smooth muscle cells (SMA‐positive). Conclusions: Results revealed that in only 5% DMSO, hSKPs could be cryopreserved for long‐term storage with considerable survival and proliferation levels, without losing multipotency.     

Viability and fertilizing capacity of cryopreserved sperm from three North American acipenseriform species: a retrospective study

Populations of sturgeon across the globe are threatened due to unregulated harvest and habitat loss, and the status varies among species across North America. Ready access to viable and functional sperm would contribute to recovery programmes for these species. In this study, we examined the motility, viability (cell membrane integrity) of cryopreserved sperm from three North American acipenseriform species and fertilizing capacity. Milt samples were collected from captive shortnose sturgeon (Acipenser brevirostrum), wild paddlefish (Polyodon spathula) and pallid sturgeon (Scaphirhynchus albus) and cryopreserved using combinations of Modified Tsvetkova’s (MT) extender, Original Tsvetkova’s extender, and modified Hanks’ balanced salt solution, along with the cryoprotectants methanol (MeOH) or dimethyl sulfoxide (DMSO). A dual‐staining technique using the fluorescent stains SYBR‐14 and propidium iodide was employed with flow cytometry to determine the percentages of spermatozoa that were viable by virtue of having intact membranes. The percentage of viable spermatozoa ranged from 5% to 12% in shortnose sturgeon, 30–59% in paddlefish, and 44–58% in pallid sturgeon. In the first experiment with shortnose sturgeon sperm, methanol allowed for higher values for dependent variables than did DMSO, and sperm viability generally correlated with post‐thaw motility. However, fertilization rate, neurulation, or hatching rates were independent from these factors. In the second experiment with shortnose sturgeon, 5% MeOH combined with MT yielded higher values for all parameters tested than the other combinations: viability was correlated with motility, fertilization rate, and hatching rate. Overall, viability and post‐thaw motility was not affected by the use of hyperosmotic extenders (OT) or cryoprotectants (DMSO), but their use decreased fertilization percentages. For paddlefish sperm (experiment 3), MT combined with 10% MeOH was clearly a good choice for cryopreservation; viability and motility results were correlated, but independent of fertilization. For pallid sturgeon sperm (experiment 4), MT with 5–10% MeOH showed significantly higher sperm quality and fertilization parameters. Membrane integrity can be used as a predictor of fertilization by cryopreserved sperm, however additional sperm quality parameters, supplementary to motility and membrane integrity, would be useful in the refining and optimizing cryopreservation protocols with acipenseriform sperm.     

Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio

Sperm cryopreservation offers potential for long-term storage of genetic resources. However, the current protocols for zebrafish Danio rerio are cumbersome and poorly reproducible. Our objective was to facilitate adoption of cryopreservation by streamlining methods from sperm collection through thawing and use. First, sperm activation was evaluated, and motility was completely inhibited when osmolality of the extender was >/=295-300mOsmol/kg. To evaluate cryoprotectant toxicity, sperm were incubated with dimethyl sulfoxide (DMSO), N,N-dimethyl acetamide (DMA), methanol, or glycerol at 5, 10, and 15% concentrations. Based on motility, DMSO, DMA, and methanol (相似文献   

Seasonal variations in semen characteristics, semen cryopreservation, estrus synchronization, and successful artificial insemination in the spotted deer (Axis axis)

Ten adult male spotted deer were monitored over a 2-year interval to determine seasonal variations in testicular size, semen characteristics and serum testosterone concentrations, and to determine if there was an association between season and type of antler. Mean (+/-S.E.M.) testicular volume (118.8+/-4.6 cm(3)), serum testosterone concentration (1.2+/-0.1 ng/mL), semen volume (4.1+/-0.6 mL), sperm concentration (338.3+/-24.9 x 10(6) mL(-1)), percentage of morphologically normal sperm (79.1+/-2.8%), and percentage of motile sperm (66.5+/-1.5%) were higher (P<0.05) in hard antler deer (peaked from March to May) than in deer with velvet antlers or in deer in which the antler has been shed. Thus, March-May was considered the physiologic breeding season for these deer; at this time, all stags had hard antlers. Furthermore, a Tris-citrate-based semen extender containing 4% glycerol and 20% egg-yolk was adequate for cryopreservation of semen. Estrus was induced with an implant containing norgestomet, timed transcervical AI was done with fresh semen, and 3 of 10 females were pregnant at 60 days, with fawns born 120 (premature), 240 and 243 days after AI. These results were considered a model for the use of assisted reproductive techniques to conserve other critically endangered deer species of India.