The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.     

Evaluation of Tris–citric acid,skim milk and sodium citrate extenders for liquid storage of Punjab Urial (Ovis vignei punjabiensis) spermatozoa

The Punjab Urial (Ovis vignei punjabiensis) is an endangered subspecie of ovidae, distributed as small scattered populations in the forest belt of the Himalayan foothills of Pakistan and in the areas enclosed by the Indus and the Jhelum rivers. The present study was conducted to evaluate the liquid storage of Punjab Urial spermatozoa in different extenders for use in future in situ conservation activities. Semen was collected by electro-ejaculation from three captive Punjab Urial rams. Suitable ejaculates of individual animals were pooled and divided into three aliquots for dilution with the experimental extenders (Tris–citric acid, skim milk and sodium citrate) at 37 °C. Extended semen was cooled from 37 °C to 5 °C in 2 h, and stored for three days at 5 °C. Sperm motility (%), viability (%; live/dead), acrosome integrity (%) and plasma membrane integrity (%) were assessed on days 1, 2 and 3 of storage. On day 1, sperm motility, viability as well as acrosome and plasma membrane integrity were similar (p > 0.05) in all three experimental extenders. On day 2, sperm motility, viability, acrosome and plasma membrane integrity were higher (p < 0.05) in Tris–citric acid extender compared to sodium citrate based extender. On day 3 of storage, the values of motility, viability and acrosome integrity were higher (p < 0.05) in Tris–citric acid extender than in skim milk and sodium citrate based extenders. In conclusion, Tris–citric acid extender appears to be a better option compared with skim milk and sodium citrate extenders for liquid storage of Punjab Urial semen.     

Effects of cryopreservation at various temperatures on the survival of kelp grouper (Epinephelus moara) embryos from fertilization with cryopreserved sperm

Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16–22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16–22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16–22 somite stage embryos survived when cooled at −25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at −25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at −140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (−196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques.     

Sperm quality and fertility of boar seminal doses after 2 days of storage: Does the type of extender really matter?

The present approach was designed to evaluate the extender effects on sperm quality and fertility of short-term refrigerated seminal doses from Landrace boars lodged in husbandry-controlled conditions. For this purpose, we analyzed the sperm quality of seminal doses diluted in short-term (Beltsville Thawing Solution) and extra-long-term (Duragen) extenders from Days 0 to 2 of storage at 17 °C during an 8-month period. Pregnancy rates and litter size were evaluated from double inseminations within an interval of 12 hours (36 and 48 hours of refrigeration) of multiparous females using seminal doses diluted in each extender type. Sperm quality was assessed from the analyses of sperm motility and kinetics, sperm viability, expressed as plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, and acrosin activity. Results indicated significant differences between the extenders in the sperm quality of seminal doses. Therefore, the seminal doses diluted in Duragen had higher percentages of progressive motile spermatozoa and membrane-intact spermatozoa than those diluted in Beltsville Thawing Solution throughout all the experimental months. Nevertheless, despite these differences in preserving the sperm quality, pregnancy rates (>90%) and litter sizes (>10 piglets born per litter) were similar between the extenders. Our results had great relevance from a practical point of view because they reported lack of an extender effect on the reproductive performance of seminal doses during short-tem storage.     

Development of extender based on soybean lecithin for its application in liquid ram semen

The soybean lecithin is used as a phospholipids source for the commercial extenders available for freezing bull semen which allows replacing the traditional membrane protective of animal origin (egg yolk). These extenders have been tested for freezing semen in various livestock species but specific adjustments cannot be made due to trade protection. The aim of the present study was to develop a soybean-based extender analyzing the optimal conditions of preparation, handling, and storage in order to optimize its use in liquid ram semen. Its effect on the quality of liquid ram semen was also studied. Different TES-Tris-Fructose-based extenders were prepared using two soybean types (S20 and S95) differentiated by their lipid composition (complex or simple, respectively). These extenders were made up in two temperatures: 20 °C (PT20) or 37 °C (PT37); centrifuged and filtered at 20 °C and stored at 15 °C or 5 °C (ST15 and ST05) for several periods (from 6 hours to 7 days). Three different concentrations of soybean (0.5%, 2%, and 3.5%) were evaluated for each extender. The amount and nature of phospholipids present in the extender were evaluated by high performance liquid chromatography (HPLC) method according to the different parameters applied in their preparation. In general, the highest quantity of phospholipids is observed in S20 extender. Centrifugation-filtration process during the extender preparation reduces by 50% the quantity of phospholipids in medium for different experiments. The quantity of phospholipids was not affected significantly by preparation temperature in S20 extender. Storage temperature affects the phospholipids present in the extender (S20 and S95) with minimum values for the storage at 5 °C. As for the storage time, both extenders (S20 and S95) showed a stable quantity of phospholipids in the course of the time, for 2 days at 15 °C and for 7 days at 5 °C. The extender obtained with a higher concentration of soybean (3.5%) showed a higher content of phospholipids under different conditions tested. Finally, sperm motility and viability in new extenders were analyzed. We observed that the sperm quality is not affected by storage temperature for S20 extender. Sperm motility was higher in S20-2% extender and control (UL). Our results suggest that a soybean lecithin extender obtained from S20 soybean at 20 °C, centrifuged and filtered, preserve the sperm motility and viability at 15 °C and 5 °C as an egg-yolk extender.     

Flow cytometric analysis from fish samples stored at low,ultra-low and cryogenic temperatures

Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (−20 °C), ultra-low (−80 °C) and cryogenic temperatures (−196 °C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (−20 °C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (−80 °C) and cryogenic (−196 °C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at −80 °C and −196 °C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation.     

Sperm cryopreservation in the spermcasting Australian flat oyster Ostrea angasi by a programmable freezing method

Cryopreservation offers long-term storage of gametes without constraint from seasonal gamete maturation, provides opportunities to improve the efficiency of breeding and genetic programs, and protects endangered species from extinction due to epidemic diseases and natural disasters. In this study, a protocol for cryopreserving sperm of the spermcasting Australian flat oyster Ostrea angasi was developed by optimizing key factors influencing the quality of cryopreserved sperm. Dimethyl sulfoxide (DMSO) was non-toxic to sperm within the concentration and duration assessed in the toxicity experiment whereas 10% methanol or a higher concentration was toxic to sperm from the exposure duration of 30 min onwards. DMSO produced higher post-thaw sperm motility among the treatments with a single cryoprotectant. The inclusion of trehalose or glucose with DMSO further increased the post-thaw sperm motility (%) and plasma membrane integrity (PMI). Sperm equilibrated for 30 min showed higher post-thaw motility and PMI than those for 10 or 50 min. Higher post-thaw sperm motility and PMI were achieved at the freezing rate of −3 °C/min than at −7 °C/min. Sperm packaged in 0.5 ml straws had a higher post-thaw motility and PMI than those packaged in 0.25 ml straws. In this study, 44.4% post-thaw sperm motility and 49.2% PMI were achieved when sperm were equilibrated in 10% DMSO +0.45 M trehalose for 30 min, packaged in 0.5 ml straws, frozen at −3 °C/min from 4 °C to −80 °C, and thawed at 40 °C for 8 s. The availability of viable cryopreserved sperm would open an option for future breeding and genetic improvement programs for the spermcasting Australian flat oyster.     

Short-term storage sperm of coho salmon (Oncorhynchus kisutch) at 4 °C: Effect of sperm: Extender dilution ratios and antioxidant butyl-hydroxytoluene (BHT) on sperm function

Short-term storage of semen is a necessary key procedure in fish; it allows maximizing the use of gametes. Nevertheless, sperm quality decreases during storage has been associated with oxidative stress damage due to an increase in reactive oxygen species (ROS) during storage. This study was designed to optimize a short-term storage protocol for Coho salmon (Oncorhynchus kisutch) spermatozoa, evaluating the effect of extender dilution and the addition of butyl-hydroxytoluene (BHT) antioxidant on sperm function parameters. In the first experiment, fresh semen was diluted in Storfish®: extender dilution (1:2 and 1:3) and a control sample undiluted and stored at 4 °C for 7-days. In both experiments motility (MO), viability and integrity of plasma membrane, mitochondrial membrane potential (MMP) and superoxide anion level (O₂⁻) were evaluated at 0, 3 and 7 days. Result shows that, 1:3 dilution maintained a higher sperm function for a longer period time. In the second experiment, spermatozoa were suspended in Storfish® (1:3) supplemented with two different concentrations of BHT (1.0 mM and 2.0 mM) and a control sample without antioxidant and stored at 4 °C for 7 days. The results demonstrated that, antioxidant-supplemented samples greater MO than control samples (P < 0.05). The viability remained >75% during storage in all groups. MMP was higher in 2.0 mM BHT compared to 1.0 mM and control (P < 0.05), in addition, this concentration reduced O₂⁻ level (P < 0.05). In conclusion, sperm: extender dilution 1:3 and adding of 2.0 mM BHT in sperm storage extender may enhance protection sperm function in Oncorhynchus kisutch against effects harmful of the oxidative stress during the in vitro storage.     

Rooster spermatozoan motility,forward progression,and fertility after storage at 25, 5, or −196 °C in various extenders

Rooster spermatozoa were stored at 25, 5, or ?196 °C in either TC199, a pyruvate-lactate mouse ova culture medium, or as undiluted semen. There was a linear decrease in percent of motile sperm during storage at 25 or 5 °C in all cases, and a curvilinear decrease with increasing storage times at ?196 °C. Percent of motile sperm present after increasing storage time suggested pyruvate-lactate is a better extender than TC199 at the three storage temperatures studied. Pullets inseminated with 1 × 10⁸ motile sperm using fresh sperm diluted in TC199 or pyruvate-lactate, or stored 24 hr at 5 or ?196 °C produced 68.7, 74.1, 20.6, and 10.8% fertile eggs, respectively. The differences in fertility between controls or between samples stored at 5 and ?196 °C were not significant. However, fertility from sperm stored at 5 and ?196 °C was significantly lower (p < .05) than both control groups. Thus, it can be concluded that TC199 or pyruvate-lactate may be used to dilute fresh rooster semen collections prior to insemination. In contrast, fertility of rooster sperm is not satisfactorily maintained after 5 or ?196 °C storage for 24 hr in a pyruvate-lactate extender.     

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 degrees C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 degrees C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.     

The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters

The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN₂). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05).The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05).In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.     

Advances in cooled semen technology.

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen.Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed.These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.     

Effect of native phosphocaseinate on the in vitro preservation of fresh semen

The fertilization capacity of goat sperm stored in milk extenders is approximately 12-24h. Long-term storage of goat sperm (up to 3 days) is desirable as it would confer greater flexibility to breeding farms. The aim of this study was to evaluate in vitro motility parameters of buck spermatozoa for up to 7 days of storage using skim milk or chemically defined extender supplemented with native phosphocaseinate (NPPC). Four experiments were conducted to determine optimum temperature (4 or 15 degrees C) and storage conditions (aerobic versus anaerobic), the effect of seminal plasma on sperm survival, the optimal concentration of NPPC and the effect of beta lactoglobulin (BL). Both skim milk and NPPC were found to be more efficient for preserving goat sperm at 4 degrees C than at 15 degrees C (P<0.01). Furthermore, when sperm was stored at 4 degrees C, no detrimental effects of seminal plasma were observed. Our results showed that motility parameters can be maintained with success until Day 4. However, NPPC-based extenders extend the in vitro survival to 7 days of storage. The optimal concentration of NPPC for the preservation of sperm cells for 4 days of storage was 81g/l and for 7 days of storage was 81 and 54g/l. No effect of the supplementation of the NPPC extender with BL was found.     

Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa

The aim of this study was to evaluate different antioxidants-supplemented freeze-dried egg yolk based extenders for the post-thawing quality and incubation resilience of goat spermatozoa. Pooled semen were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in control and antoxidant supplemented freeze-dried extenders (methionine, cysteamine and butylated hydroxytoluene). Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Membrane lipid peroxidation status was also analyzed using the malondialdehyde (MDA) concentration. In the study, antioxidant supplemented freeze-dried egg yolk based extenders have beneficial effect on goat sperm parameters. In addition, we achieved a higher quality in post thawed goat semen even after 6 h incubation when the extender was supplemented by 5 mM BHT or cysteamine.     

海藻糖对猪精子冷冻真空干燥保存效果的影响

猪精子经冷冻干燥后,在光学显微镜和电子显微镜下观察其超微结构,并借助辅助生殖技术将其注入猪卵母细胞后,进一步观察受精卵的发育情况。结果表明：海藻糖组雄原核形成率 (68.52％)、卵裂率 (59.17％) 和囊胚率 (19.16％) 优于EDTA组 (64.59％、56.26％和15.62％) 和对照组 (35.36％、52.33％和8.60％) (P＜0.05);海藻糖组的冷冻真空干燥猪精子分别在4℃下保存60、120、180 d,雄原核形成率、卵裂率和囊胚率均无显著差异 (P＞0.05);海藻糖组的冷冻真空干燥猪精子复水化后孵育1 h和2 h,卵裂率、卵裂率和囊胚率均差异显著 (P＜0.05);海藻糖处理组与EDTA处理组中的冷冻真空干燥猪精子分别在4℃和?20℃下保存后各处理组间精子形态差异不显著 (P＞0.05);海藻糖组中B级冷冻真空干燥精子百分数显著多于EDTA处理组 (P＜0.05)。超微结构分析表明,冷冻真空干燥猪精子的损伤主要表现在顶体和颈部的肿胀与缺损、尾部断裂。     

Effect of different glycerol concentrations on phosphatidylserine translocation and mitochondrial membrane potential in chilled boar spermatozoa

Boar spermatozoa are extremely sensitive to low temperatures and the cryopreservation causes dramatic changes in sperm survivability, but it is not clear which part of the cryopreservation process affects the most. The aim of this work was to assess early events of apoptotic changes as damage indicators in boar sperm cooled to 5 °C and exposed to different glycerol (GLY) concentrations. For this purpose, progressive sperm motility (CASA), plasmatic and acrosome membranes integrity (CFDA/PI; phase contrast), plasma membrane functionality (HOS), phosphatidylserine translocation (Annexin-V/FITC) and reduction of mitochondrial membrane potential (Ψm) (JC-10) were carried out at 37 °C, 17 °C and 5 °C in eight boar sperm pools. Afterwards, three aliquots were diluted in different freezing extenders (control: 0% GLY; A: 2% GLY and B: 3% GLY); sperm quality and early apoptotic changes were assessed. Motility was negatively affected during cooling to 5 °C. Furthermore, plasma membrane functionality was the most affected by cooling. The number of necrotic cells was higher at 5 °C. However, no differences were observed in phosphatidylserine translocation. The extender with 3% GLY at 5 °C presented better Ψm than 0 and 2% GLY. Based on this analysis, boar sperm cooling to 5 °C does not modify the rate of early apoptotic changes, although alterations in the Ѱm were evident.     

Storage stability and sourdough acidification kinetic of freeze-dried Lactobacillus curvatus N19 under optimized cryoprotectant formulation

In this study, the response surface methodology was used to optimize the cryoprotective agent (skimmed milk powder, lactose and sucrose) formulation for enhancing the viability of Lactobacillus curvatus N19 during freeze-drying and storage stability of cells freeze-dried by using optimum formulation was evaluated. Our results showed that the most significant cryoprotective agent influencing the viability of L. curvatus N19 to freezing and freeze-drying was sucrose and skim milk, respectively. The optimal formulation of cryoprotective agents was 20 g/100 mL skim milk, 3.57 g/100 mL lactose and 10 g/100 mL sucrose. Using the optimum formulation during freeze-drying, the cell survival was found more than 98%. Under the optimal conditions, although only storage of the cells at 4 °C for 6 month retained the maximum stability (8.85 log cfu/g), the employed protectant matrix showed promising results at 25 °C (7.89 log cfu/g). The storage stability of cells under optimized conditions was predicted by accelerated storage test, which was demonstrated that the inactivation rate constant of the freeze-dried L. curvatus N19 powder was 9.74 × 10⁻⁶ 1/d for 4 °C and 2.08 × 10⁻³ 1/d for 25 °C. The loss of specific acidification activity after the storage at 4 and 25 °C was determined.     

Mink semen studies: I. Liquid preservation and prospect of freezing spermatozoa

Using 22 males, 41 semen samples were collected from the vagina of mink by means of a plastic tubing attached to a 1 ml syringe. Subsamples of vaginal semen were diluted in 4 different extenders, viz., tris (tris, citric acid, glycine, fructose, glycerol and egg yolk), PVP (tris extender with polyvinyl pyrrolidone and caproic acid), milk (boiled and filtered milk with glycerol) and sodium citrate. The extended semen samples were stored at 23, 5 and −196°C for varying periods and evaluated for % motile spermatozoa. In the tris extender storage for 3 days at 5°C or for 2 days at 23°C reduced the number of spermatozoa by more than 50%. When milk was used as the extender, the motility decreased from an initial value of 68% to 10% after 5 days of 5°C and to 8% after 4 days at 23°C. The PVP extender was not suitable for storage at any temperature. After being frozen at −196°C for 2 hr, the motility ranged from 3–10% in the tris extender and was zero in milk and PVP extenders. Prolonged storage for 7 days in tris extender reduced the motility to 1–7%.     

Preliminary study on the cryopreservation of tropical bagrid catfish (Mystus nemurus) spermatozoa; the effect of extender and cryoprotectant on the motility after short-term storage

The effects of different extenders, and cryoprotectants on the motility of tropical bagrid catfish (Mystus nemurus) spermatozoa were evaluated after short-term storage. Three extenders, physiological saline, Ringer or saline at three levels of sperm to extender dilutions (1:20, 1:30, or 1:40) and four cryoprotectants (DMSO, ethanol, glycerol or methanol) at three concentrations (5, 10, or 15%) were examined in two separate experiments. In the first experiment, milt was suspended in the respective extender at the three milt to extender dilution ratios in two sets of tubes. Extended milt in the first set of tubes was stored at -4 degrees C, and motility assessed after 24h, while the second set was kept at 23 degrees C and sperm motility was assessed immediately and at 30-min intervals thereafter. Ringer retained sperm motility better than the other extenders at all dilution levels at temperatures of 23 and -4 degrees C respectively. At 23 degrees C, the sperm motility was almost completely lost after 150 min except for those in Ringer at 1:20 dilution level which still had a motility of 18% (compared to those kept at -4 degrees C for 24, which had motility from 39 to 71%, regardless of extender). In the second experiment, various cryoprotectants were added to solutions of milt (that was diluted in Ringer at 1:20 ratio and cryopreserved in liquid nitrogen for 15 days). Sperm cryopreserved in 10% methanol had the highest motility (58%) compared with those in the other cryoprotectants at all concentrations.     

Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 x 10(6) sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4 degrees C). Three cooling rates (-1.0, -0.3 and -0.1 degrees C/min) were also investigated at the lowest storage temperature (4 degrees C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4 degrees C than at 35 or 22 degrees C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4 degrees C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (-1.0 and -0.3 degrees C) than at the slow cooling rate (-0.1 degrees C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4 degrees C. Additional studies are needed to evaluate the fertility of semen stored in this manner.