Preliminary Trial: Motility Comparisons of a Unique Freezing Technology (UFT) to Liquid Nitrogen Mist Methodology for Cryopreservation of Porcine Spermatozoa

The motility outcomes of boar semen frozen with newly developed freezing techniques using a new unique freezing technology (UFT) compared with traditional liquid nitrogen methodology were investigated with the intent of improving current fertility outcomes using semen. The UFT is an electronically controlled cooling chamber that houses an organic fluid bath that can be maintained at temperatures below 0 degrees C without solidifying to freeze samples. Four ejaculates from four different boars were collected for this trial. Samples were handled consistently during the pre- and post-freeze processing. From each ejaculate, samples were separated into eight cryopreservation treatment groups, six UFT variations and two control liquid nitrogen groups, immediately before freezing, in replicates of two. After the initial cryopreservation was complete, all samples were stored in liquid nitrogen for at least 48 h. Post-thaw motilities and original motility return percentages were assessed on a random, individual-sample basis. After the initial evaluations, samples from two boars were recollected and frozen using the UFT for breeding purposes. Four sows were bred with the UFT frozen semen to confirm fertility capability. When assessing the individual UFT techniques, all of six UFT techniques had improved post-thaw motilities. However, treatments F (micro = 29%, return micro = 37%) and J (micro = 27%, return micro = 34%) showed the highest statistical improvement for post-thaw (p < 0.05) and original motility percent returns (p < 0.05) when compared with either the control cryo-tube (micro = 15%, return micro = 19%) or straw groups (micro = 12%, return micro = 16%). The UFT semen had a 50% conception rate, with an average of seven piglets from the sows that farrowed. Our preliminary data suggest a higher motility return with a slower pre-freeze phase below the freezing point before the acceleration to liquid nitrogen temperatures. The preliminary data suggest that the UFT could be utilized as a potential cryopreservation option for boar semen.     

Preliminary comparisons of a unique freezing technology to traditional cryopreservation methodology of equine spermatozoa

With recent large-breed organization acceptance, the use of frozen semen is gaining more attention in the equine industry. However, cryopreserved stallion semen is commonly associated with poor quality and decreased pregnancy rates as compared with those produced during normal mating or with cooled semen techniques. Therefore our objective was to investigate a new unique freezing technique (UFT) with the intent of improving fertility outcomes. A series of experiments tested the UFT compared with traditional liquid nitrogen methodology in combination with influence of the extenders and stallions used. In Experiment 1, post-thaw motility results of UFT variations were compared with those from liquid nitrogen methods. The averaged post-thaw motility percentages of the 4 UFT treatments were similar when compared with the traditional liquid nitrogen control group (P = .845). In Experiment 2, 2 egg-yolk–based freezing extenders, Biladyl AB intended for bovine samples and Freezing Medium Test Yolk Buffer used for human samples, were compared. A significant difference in the average post-thaw motilities was found between Biladyl AB (17%) and Freezing Medium Test Yolk Buffer (25%) (P < .002). In the third experiment, we compared variability among stallions using the UFT with the intention of creating a more consistent outcome. Post-thaw motilities and percent of original motility returns among the 4 stallions were significantly different (P < .001). In a field trial using shipped semen from a regional stallion station, the UFT demonstrated very promising results.In conclusion, the UFT may potentially be used as an alterative freezing method to replace current liquid nitrogen methodology. However, further investigation is needed to refine techniques.     

Comparative study of different methods for dog semen cryopreservation and testing under clinical conditions

The extenders and freezing rates from three different freezing protocols were combined and compared to each other in order to study the post-thawing acrosome integrity and fertility of frozen dog sperm. A commercial bovine TRIS-base extender (TRILADYL) and two self-made canine semen extenders (Norwegian and Dutch) were combined with a conventional bovine and two canine freezing regimes, and acrosome integrity of frozen/thawed spermatozoa was assessed by fluorescein isothiocyanate conjugated peanut agglutinin staining (FITC-PNA). Differences between freezing/thawing protocols were reflected in the proportion of cells with acrosomal damage and not based on motility results. It was concluded that during dog semen cryopreservation extenders had less influence on the post-thawing sperm quality than did the freezing rates. The optimal extender/freezing rate combination (TRILADYL/Norwegian) was used in the clinical practice to evaluate the fertility of frozen sperm administered by intrauterine insemination using a surgical approach. The pregnancy rate was 57% (4/7), but the average litter size was low (2.8). This may have been due to the insufficient sperm numbers contained in an insemination dose and/or to the incorrect timing of artificial insemination (AI). The final conclusion is that the commercial bovine extender is useful for freezing dog semen, and the TRILADYL/Norwegian freezing protocol is recommended as the most advantageous combination for the freezing of canine semen in the clinical practice.     

辅酶Q10对绒山羊精液冷冻保存效果的影响

旨在探讨辅酶Q10对绒山羊精液冷冻保存效果的影响。利用添加不同浓度辅酶Q10(4、40、400?滋g/mL)的精液冷冻稀释液对绒山羊精液样本进行冷冻保存,待冷冻精液解冻后,采用流式细胞仪和计算机辅助精液分析系统(CASAS)分别检测不同精液样本的精子活率、质膜完整率、顶体完整率、DNA完整率、线粒体膜电位和细胞内ROS水平。结果表明,当冷冻稀释液中添加浓度为40μg/mL辅酶Q10时,经历冷冻—解冻过程的绒山羊精液样本的精子活率、质膜完整率、顶体完整率均显著高于对照组(P<0.05);在冷冻稀释液中添加浓度为40μg/mL或400μg/mL的辅酶Q10均能显著提高线粒体膜电位并降低细胞内ROS水平(P<0.05)。综上所述,在冷冻稀释液中添加40μg/mL的辅酶Q10能够显著提高绒山羊精子抗氧化能力和冷冻保存效果。     

牛精子冷冻损伤及其改善方法

牛精液冷冻与人工授精技术相结合，在牛品种改良、保护优良种质资源中发挥着重要作用。尽管冷冻后部分牛精子活力高达60%以上，但是冷冻后精子受损现象却普遍存在，尤其是受精能力与鲜精相比显著降低。作者详细介绍了冷冻-解冻过程对牛精子造成的主要损伤，包括精子的形态完整性、活力及遗传物质的改变等；阐述了冷冻造成精子损伤的主要原因，即细胞内冰晶形成和氧化应激反应及其产生的可能机制；并且对目前常用的提高冷冻精子质量的方法，如添加冷冻保护剂、优化冷冻程序以及添加抗氧化剂等进行了详细地综述；提出了在冷冻精液研究方面值得探索的问题，以期为家畜精液冷冻保存技术的进一步优化提供理论依据。     

The effect of Curcuma longa extracted (curcumin) on the quality of cryopreserved boar semen

The aim of this study was to determine the optimal concentration of curcumin needed for cryopreservation of boar semen. Semen samples (n = 9) were collected from nine Duroc boars which having proven fertility were used for routine artificial insemination. Semen samples were collected and divided into six groups (groups A‐F) according to various concentrations of curcumin in freezing extender (i.e. 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively). The semen was frozen by traditional liquid nitrogen vapor method and stored at ?196°C in the liquid nitrogen tank. After storage, frozen semen samples were thawed at 50°C for 12 s and evaluated for progressive motility, viability and acrosome integrity. The present results indicated that the addition of curcumin at 0.25 (group C) or 0.50 mmol/L curcumin (group D) yielded the higher percentage of progressive motility (33.3 and 36.1%, respectively) (P < 0.001). A significantly higher percentage of acrosome integrity was found in groups B (29.7%), C (31.1%) and D (30.2%) than in the other groups (P < 0.01). However, there was no significant difference in percentage of viability among groups. In conclusion, addition to the freezing extender of curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen.     

Effects of Repeated Partial Thaw and Refreeze on Post-Thaw Parameters of Stallion Semen Cryopreserved in Cryovials

The current study evaluated post-thaw semen parameters of stallion semen cryopreserved in cryovials and subjected to multiple partial thaw-refreeze cycles. Five fertile stallions were collected twice, and ejaculates were analyzed for concentration, percent membrane integrity, motility, morphology, and sperm chromatin structure (SCSA). Semen processed with freezing extender from each ejaculate was cryopreserved in both 1.2-mL cryovials and 0.5-mL straws. Cryovials were subjected to eight subsequent partial thaw-refreeze cycles. Cryovials were warmed for approximately 30 seconds; then, a sample of cryopreserved semen was removed with a 16-gauge needle, and the cryovial was immediately refrozen in liquid nitrogen. A piece of 0.5-mL straw cut under liquid nitrogen from the same stallion and ejaculate was thawed alongside each cryovial to serve as a control. Thawed samples were analyzed for percent membrane integrity, motility, and SCSA. Post-thaw parameters of motility and membrane integrity were analyzed by one-way or two-way analysis of variance with repeated measures when appropriate. The SCSA data were analyzed using a mixed regression model. Post-thaw motility and percentage of intact sperm were significantly lower when sperm was cryopreserved in cryovials compared to straws. However, these parameters may remain adequate for use in assisted reproductive techniques (ARTs) such as intracytoplasmic sperm injection through all cryovial thaws. Additionally, DNA denaturability was not affected by semen packaging method and was only affected by thaw number, increasing at post-thaws 5 and 6. This technique may offer a unique approach for cryopreservation and utilization of stallion sperm for ARTs in the future.     

Influence of Semen Storage and Cryoprotectant on Post-thaw Viability and Fertility of Stallion Spermatozoa

The aim of the current study was to verify that stallion spermatozoa could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryopreservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio^® extender containing one of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5°C for 20 minutes, then frozen in nitrogen vapor. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty-three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine semen for 24 hours before freezing, while maintaining sperm viability and fertility, is possible.     

Initial cooling time before freezing affects post‐thaw quality and reproductive performance of rabbit semen

The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post‐thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris‐citrate‐glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post‐thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms.     

Effect of Ascorbic Acid Concentrations,Methods of Cooling and Freezing on Boer Goat Semen Cryopreservation

To improve the Boer goat semen quality during cryopreservation process, three experiments were carried out to investigate the effect of (i) different concentration of ascorbic acid supplementation (ii) rate of cooling with chilled semen characteristics and (iii) method of freezing on post‐thaw Boer goat sperm using Tris‐based extender. Ascorbic acid at 8.5 mg/ml improved the sperm parameters (motility, integrity of membrane and acrosome, morphology and viability), compared to control in cooled samples (p < 0.05). With regard to other concentrations and post‐thawed parameters, ascorbic acid at 2.5–8.5 mg/ml led to higher percentages of sperm motility and integrities of membrane and acrosome when compared to control (p < 0.05). Slow cooling rises to higher percentages of sperm motility, acrosome integrity and viability, in comparison with fast cooling, in terms of cooled and frozen samples (p < 0.05). Programmable freezing method produced the higher percentages of sperm motility, integrities of membrane and acrosome and viability when compared to the freezing method of polystyrene box during goat sperm freezing (p < 0.05). In conclusion, chilled and post‐thawed sperm quality of Boer goat was improved when a Tris‐based extender supplemented with ascorbic acid was used at stages of different cooling rates and freezing methods.     

Comparison of two different centrifugation extenders for preservation of frozen equine semen

This study compares a commercial semen extender (control group) to ultra high temperature (UHT) skimmed milk (treatment group) used during centrifugation for subsequent cryopreservation of equine semen. Following post‐thawing of semen samples parameters measured included motility, sperm motion kinetics (using computerised assisted semen analysis) as well as acrosome and plasmatic membrane integrity (using fluorescent dyes). After collection and analysis, the sperm‐rich fraction was divided and diluted with either: control (1:1 dilution in a skimmed milk‐glucose extender) or treatment (1:1 dilution in UHT skimmed milk). The milk used in this experiment was of the same source, commercial brand, of only one lot. After dilution, samples were subjected to centrifugation at 600 g for 10 min and sperm pellets were resuspended in a freezing extender to a concentration of 200 × 10⁶ cells/ml. Aliquots were packed into 0.5 ml straws placed in a stainless steel support and kept inside the refrigerator (5°C) for 20 min. Subsequently, these straws were placed at a height of 6 cm over liquid nitrogen for 20 min in an isotherm box. No significant differences were observed in total sperm motility (42.71 vs. 38.29%), progressive sperm motility (12.29 vs. 7.86%), plasma membrane integrity (53.43 vs. 60.14%) or acrosomal membrane integrity (93.29 vs. 93.71%) with a P>0.05 calculated between the control and the treatment groups, respectively. Considering that UHT skimmed milk has a lower cost than the commercial semen extender, this could be an option used during the centrifugation protocol to decrease the expense of the equine semen cryopreservation process and increase shelf life.     

Optimization of glycerol concentration and freezing rate in the cryopreservation of ejaculate from brown bear (Ursus arctos)

In order to establish a semen bank for the endangered Cantabrian brown bear, we tested five glycerol concentrations and three freezing rates for electroejaculated semen. Electroejaculation was performed on nine males. Semen was diluted in TES-Tris-Fructose (20% egg yolk, 2% EDTA, 1% Equex) with 2%, 4%, 6%, 8% or 10% glycerol and frozen at -10, -20 or -40°C/min. Before and after cryopreservation, samples were analysed for motility (CASA), viability and acrosomal status (flow cytometry). Pre-freezing results showed that glycerol concentration had no significant effect on total motility or progressive motility, but it significantly decreased VCL, ALH, viability and acrosomal status (p < 0.05). After thawing, sperm motility was higher at extender with 4%, 6% and 8% glycerol, but only at 4% and 6% glycerol for viability and acrosomal status. For 4% and 6% glycerol, freezing rates did not have significant effects. The curve fitting gave an estimate of the optimal glycerol concentration, with all the optimal values for every parameter between 6% and 7% glycerol falling. We propose using 6% glycerol and a freezing velocity of -20°C/min for freezing brown bear ejaculated spermatozoa.     

Influence of Cool Storage before Freezing on the Quality of Frozen–Thawed Semen Samples in Dogs

The aim of this study was to determinate the semen quality of frozen–thawed samples that were chilled for up to 2 days before freezing. The ejaculates (n = 18) from six dogs were collected, pooled and divided into six aliquots. The first aliquot (C, control) was frozen in liquid nitrogen using a conventional protocol to reach a final concentration of 100 × 10⁶ spermatozoa/ml, 20% egg yolk and 5% glycerol. The remaining five aliquots were diluted with a chilled extender (Tris‐glucose and 20% egg yolk) and cooled at 4°C as follows: R1, the semen was cooled for 1 h; R6, the semen was cooled for 6 h; R12, the semen was cooled for 12 h; R24, the semen was cooled for 24 h and R48, the semen was cooled for 48 h. After the chilling period, a second extender was added (Tris‐glucose, 20% egg yolk, 10% glycerol and Equex at 1%) to reach a final composition similar to aliquot C, and then, the semen samples (R1, R6, R12, R24 and R48) were frozen in liquid nitrogen. The post‐thaw sperm quality was assessed in 30 straws from each experimental group. After freezing–thawing, the total sperm motility (approximately 60–70%) in the semen chilled for up to 48 h did not show any differences from the samples frozen by the conventional cryopreservation method (63.2%). No significant differences were detected in the percentages of abnormal sperm cells among the fresh semen, the control group and the frozen samples after the different cooling times. Finally, the post‐thaw percentages of damaged acrosomes showed a very uniform distribution, with mean values ranging between 7% and 10.5%. The results clearly demonstrated that cooling the semen up to 48 h before freezing did not produce a decrease in the semen quality when was compared with semen frozen by a traditional procedure.     

Effect of Docosahexaenoic Acid on Quality of Cryopreserved Boar Semen in Different Breeds

During the cryopreservation process, the level of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), in the sperm plasma membrane decreases significantly because of lipid peroxidation, which may contribute to sperm loss quality (i.e. fertility) of frozen–thawed semen. The aim of this study was to investigate the effect of supplementation of DHA (fish oil) in freezing extender II on frozen–thawed semen quality. Semen from 20 boars of proven motility and morphology, were used in this study. Boar semen was split into four groups, in which the lactose–egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various levels of fish oil to reach DHA level of 1X (group I, control, no added fish oil), 6X (group II), 12X (group III) and 18X (group IV). Semen solutions were frozen by using a controlled rate freezer. After cryopreservation, frozen semen was thawed and evaluated for progressive motility, viability by using SYBR‐14/Ethidiumhomodimer‐1 (EthD‐1) staining and acrosome integrity by using FITC‐PNA/EthD‐1 staining. There was a significantly higher (p < 0.001) percentage of progressive motility, viability and acrosome integrity in DHA (fish oil) supplemented groups than control group. Generally, there seemed to be a dose‐dependent effect of DHA, with the highest percentage of progressive motility, viability and acrosome integrity in group‐III. In conclusion, supplementation of the LEY extender with DHA by adding fish oil was effective for freezing boar semen as it resulted in higher post‐thaw plasma membrane integrity and progressive motility.     

Directional Freezing of Equine Semen in Large Volumes

Despite its potential impact on the horse industry, sperm cryopreservation is not an established technology throughout the industry, for a number of reasons that include a reduction in pregnancy rate and increased cost per pregnancy. We have evaluated a novel directional freezing technique, based on a multi-thermal gradient (MTG), by comparing it with the conventional, controlled-rate cryopreservation method (CRCM). Ninety-seven ejaculates with > or =50% motility, collected from 31 stallions were each divided into two parts and subsequently frozen by either MTG or CRCM. Frozen samples were then stored in liquid nitrogen until thawing. The two treatments were evaluated by three methods: progressive linear motility (PLM), viability stain and hypoosmotic swelling (HOS) test. High correlation was found between the three evaluation methods for all post-thaw samples. Eighty-eight per cent of the ejaculates frozen by MTG had post-thaw PLM > or =35%, whereas only 59% of the ejaculates frozen by CRCM had such motility. Post-thaw evaluations of samples frozen by MTG and CRCM were: PLM - 50.2 +/- 1.5% and 37.4 +/- 1.5%, respectively; viability - 53.6 +/- 1.5% and 39.5 +/- 1.4%, respectively; membrane integrity, as evaluated by HOS - 36.2 +/- 1.3% and 26.5 +/- 1.1%, respectively. The differences according to all the evaluation methods were highly significant (p < 0.001), and the results indicate that freezing stallion semen by MTG is superior to CRCM.     

种牛精液冷冻和保存效果研究进展

随着养牛业的高速发展,牛精液液体保存技术的研究也在逐步深入。人工授精技术降低了养殖成本,加速了肉牛的繁殖改良,同时还促进了育种工作的进程,合理有效的使用人工授精技术能够提高牛的饲养效益。与此同时,优良的种牛精液品质也是改良的关键,有实验结果表明：遗传、气候环境、饲养管理等因素会影响牛的精液品质[1],同时强制运动也是决定种牛精液品质的关键环节[2]。合理的精液冷冻与保存不仅能发挥优良的精液品质,同时也为人工授精技术的发展提供保障。本文从牛精液新型冷冻保护剂和损伤修复、牛精液冷冻保存添加剂、牛冷冻精在人工受精中的应用、冷冻保存的发展前景等方面总结了我国牛精液冷冻保存技术的研究和发展,以期对今后种牛精液冷冻和保存技术发展有所帮助、提供参考。     

Effects of liquid nitrogen vapor sensitization conditions on the quality of frozen-thawed dog spermatozoa

The freezing conditions for preparation of frozen canine semen by the plunging method were investigated with regard to the period of sensitization in liquid nitrogen (LN2) vapor and the height from LN2, and the semen qualities after thawing were compared with those of canine semen prepared by the simple freezer method previously reported by us. In the plunging method, 9 semen straws were prepared under the same conditions, horizontally kept at 5, 7, and 10 cm above the LN2 surface in a styrene foam box for 5, 10, and 15 min, and then plunged into LN2. The semen qualities immediately after thawing were high in the 7 cm/10 min (cooling rate: -4 to -22 degrees C/min) and 10 cm/15 min groups (cooling rate: -6 to -10 degrees C/min). On comparison of frozen semen prepared by the plunging method (7 cm/10 min) with frozen semen prepared by the simple freezer method, sperm motility and viability were significantly higher for the frozen semen prepared by the plunging method. The cooling rate in freezing was higher for the simple freezer method (cooling rate: -6 to -50.9 degrees C/min) than the plunging method. Based on these findings, horizontal placement of canine semen straws above LN2 to reduce the temperature at a slow cooling rate of about -10 degrees C/min, followed by plunging into LN2 after sensitization for 10-15 min, provides good semen qualities after thawing.     

A comparison of duck and chicken egg yolk for cryopreservation of stallion sperm

OBJECTIVE: Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. DESIGN: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after thawing. Morphology data were also collected at 0 and 1 hr post thaw. RESULTS: Total and forward progressive motility were higher when the sperm were frozen in the presence of duck rather than chicken egg yolk. Furthermore, the total and forward progressive motility and percentage of morphologically normal sperm were higher when frozen at a concentration of 200 than 20 x 10(6)/mL. CONCLUSION: The results of this study demonstrate that the motility parameters of stallion sperm are improved when the semen is frozen in lactose EDTA extender supplemented with duck egg yolk rather than chicken egg yolk. Moreover, sperm motility and the percentage of morphologically normal sperm were higher after freezing at a concentration of 200 x 10(6)/ml rather than 20 x 10(6)/ml.     

Factors influencing boar sperm cryosurvival

Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that 70% of total variance observed in postthaw sperm quality variables among ejaculates was explained by boar. This indicates that boar is the most important (P < 0.001) factor explaining the variability among ejaculates in sperm cryosurvival, with most (14 of the 15 boars in Exp. 3) showing consistent (P > 0.05) sperm cryosurvival over time.     

The mechanism by which mouse spermatozoa are injured during freezing

To improve the cryopreservation protocol for mouse sperm, we attempted to estimate the type and extent of cryoinjury at various steps of the process. First, we demonstrated that mouse sperm are sensitive to chilling at -15 C and that the sensitivity is dependent on the length of exposure. To estimate cryoinjuries, sperm suspensions were ice-seeded at -5 or -15 C, frozen with liquid nitrogen (LN(2)) gas and then frozen in LN(2). In one experiment, sperm seeded at -5 C were cooled slowly to -15 C before deep freezing. At various steps of the cryopreservation process, the sperm were warmed and their viability was assessed based on motility and the integrities of the plasma membrane and acrosome. The motility of frozen-thawed sperm was higher on seeding at -5 C (28%) than at -15 C (9%). The motility did not decrease when the sample was transferred from LN(2) gas to LN(2). To estimate cryoinjury of sperm, we presumed the viability of frozen sperm to be decreased by chilling, hypertonic stress and formation of intracellular ice. When the sperm suspension was cooled and seeded at -5 C, the motility decreased by 25% due to hypertonic stress. When the sperm were cooled in LN(2) gas, the motility decreased by 17% with the formation of intracellular ice. When the sperm were cooled to -15 C, the motility decreased by 51% from chilling. After seeding, the motility decreased by 18% due to formation of intracellular ice and by 7% due to hypertonic stress. Considering the results, it would be preferable to seed samples at a higher temperature to prevent intracellular ice from forming and to cool seeded samples rapidly enough to minimize chilling injury and hypertonic stress, but not too rapidly to allow intracellular ice to form.