Low glucose and high pyruvate reduce the production of 2-oxoaldehydes, improving mitochondrial efficiency, redox regulation, and stallion sperm function†.

Energy metabolism in spermatozoa is complex and involves the metabolism of carbohydrate fatty acids and amino acids. The ATP produced in the electron transport chain in the mitochondria appears to be crucial for both sperm motility and maintaining viability, whereas glycolytic enzymes in the flagella may contribute to ATP production to sustain motility and velocity. Stallion spermatozoa seemingly use diverse metabolic strategies, and in this regard, a study of the metabolic proteome showed that Gene Ontology terms and Reactome pathways related to pyruvate metabolism and the Krebs cycle were predominant. Following this, the hypothesis that low glucose concentrations can provide sufficient support for motility and velocity, and thus glucose concentration can be significantly reduced in the medium, was tested. Aliquots of stallion semen in four different media were stored for 48 h at 18°C; a commercial extender containing 67 mM glucose was used as a control. Stallion spermatozoa stored in media with low glucose (1 mM) and high pyruvate (10 mM) (LG-HP) sustained better motility and velocities than those stored in the commercial extender formulated with very high glucose (61.7 ± 1.2% in INRA 96 vs 76.2 ± 1.0% in LG-HP media after 48 h of incubation at 18°C; P < 0.0001). Moreover, mitochondrial activity was superior in LG-HP extenders (24.1 ± 1.8% in INRA 96 vs 51.1 ± 0.7% in LG-HP of spermatozoa with active mitochondria after 48 h of storage at 18°C; P < 0.0001). Low glucose concentrations may permit more efficient sperm metabolism and redox regulation when substrates for an efficient tricarboxylic acid cycle are provided. The improvement seen using low glucose extenders is due to reductions in the levels of glyoxal and methylglyoxal, 2-oxoaldehydes formed during glycolysis; these compounds are potent electrophiles able to react with proteins, lipids, and DNA, causing sperm damage.

Spotted wolffish (Anarhichas minor) sperm cryopreservation in 5-mL cryovials.

In spotted wolffish Anarhichas minor aquaculture, cryopreservation is used to secure sperm availability throughout the entire spawning season. Under current protocols, sperm is cryopreserved in 0.5-mL straws. This implies thawing a considerable number of straws for insemination with cryopreserved sperm. In this work, we scale up the spotted wolffish sperm cryopreservation procedure through the development of a protocol for sperm cryopreservation in 5-mL cryovials. Different freezing (distances from the liquid nitrogen surface) and thawing rates were tested. The best results were obtained with cryovials frozen at a distance of 1.5 cm from the liquid nitrogen surface and thawed either at 15 or 10 °C for 4 and 6 min, respectively. Under these conditions, similar percentage of motile cells, sperm velocity and percentage of viable cells were obtained in comparison with the sperm cryopreserved in the traditional 0.5-mL straws. This protocol will facilitate the process of insemination with cryopreserved sperm in the spotted wolffish hatcheries.

Micro-quantity straw as a carrier for cryopreservation of oligozoospermic semen samples: Effects of storage times and cryoprotectant.

Application of an appropriate freezing carrier is crucial for improving post-thaw recovery of oligozoospermic samples. In this study, our purpose is developing a user-friendly, easy handling and close micro-quantity (MQ) straw along with different freezing media, for cryopreservation of oligozoospermic samples. Twenty oligozoospermic semen samples were collected and mixed with glycerol egg yolk citrate (GEYC) or Spermfreeze® (SPF) medium. The mixture was loaded into MQ straws, sealed and stored in liquid nitrogen (LN) vapor. After freezing, the straws were transferred into cryotube and plunged into LN. Post-thawed sperm parameters including motion characteristics, viability, membrane and DNA integrity were evaluated one and three months after cryopreservation. The post-thawed sperm parameters were significantly reduced in GEYC and SPF medium compared to fresh samples. No statistically significant differences were seen in sperm characteristics between the two storage times (i.e. month 1 vs. month 3). Furthermore, GEYC medium yielded higher motility, viability and membrane integrity compared to SPF at both storage time-points. Sperm DNA integrity was also improved in GEYC group compared to SPF after 1 month of storage. The findings of our study showed that application of MQ straw along with GEYC, as the cryoprotectant, was beneficial for cryopreservation of low count semen samples.

Preclinical evaluation of a new cryopreservation container for a limited number of human spermatozoa.

The aim of this study was to develop a new container for cryopreservation of a limited number of spermatozoa. To evaluate the efficacy and safety of this new container, we performed preclinical evaluations using human sperm or mouse oocytes and sperm. First, using human sperm that was frozen and then thawed, we demonstrated that the sperm recovery rate using the new container was 96.7% (58/60), which was significantly higher (P < 0.05) than the recovery rate of 21.2% (11/52) when using the Cryotop®. Sperm motility rates were 19.2% (10/52) using the Cryotop® and 35.0% (21/60) using the new container. Second, murine epididymal spermatozoa were divided into three groups: fresh spermatozoa, spermatozoa frozen using a straw, and spermatozoa frozen using the new container. Sperm motility, sperm membrane and DNA integrity, in vitro development of fertilized eggs, and offspring development after embryo transfer were assessed. The motility of freeze-thawed sperm was lower in spermatozoa that were frozen using the new container than in fresh spermatozoa or those that were frozen using a straw. After intracytoplasmic sperm injection, the survival rate was 96.7% (145/150), the 2-cell development rate was 90.3% (131/145), and the blastocyst development rate was 77.2% (112/145), when using the new container. There were no differences in the sperm membrane, DNA integrity, or in the embryo development rates to the blastocyst stage among the different frozen groups. Six offspring were derived from spermatozoa freeze-thawed in the new container, and they developed normally. Thus, the new container allows easy handling of a small number of sperms and minimizes sperm loss during cryopreservation.

Does supplementation of sperm freezing/thawing media with Ceratonia siliqua improve detrimental effect of cryopreservation on sperm parameters and chromatin quality in normozoospermic specimens?

Human sperm banking is an important procedure in the assisted reproductive technique centers. It entails sperm damage. The aim of this study was to investigate beneficial effect of Ceratonia siliqua (C. siliqua) supplement in freezing/thawing media on post thaw sperm parameters and sperm chromatin quality in normozoospermic samples. Forty normozoospermic specimens were included in this prospective study. Each sample was divided into ten groups. In groups one to five, 0 (as control group) 5, 10, 20 and 30 µg/ml C. siliqua were added to freezing medium and in groups six to ten, similar concentration of C. siliqua were added to thawing medium for 30 min incubation. Sperm concentration, progressive motility, normal morphology, viability, aniline blue (AB), toluidine blue (TB) and sperm chromatin dispersion (SCD) staining tests were evaluated before vitrification and after thawing. The results showed that 10 and 20 µg/ml supplementation of C. siliqua in freezing/thawing media significantly increased progressive motility, normal morphology and viability of sperm (p < 0.05) as well as decreased AB, TB and SCD (p < 0.05). Also, 20 µg/ml had significantly higher improvement compared to 10 µg/ml C. siliqua (p < 0.05). The present study showed that C. siliqua supplemented freezing/thawing media can improve sperm quality of normozoospermic samples after freezing/thawing.

[Self-made metal freezing plate for cryopreservation of human epididymal sperm: An experimental study].

OBJECTIVE: To study the effect of a new human sperm freezing method on the sperm recovery rate and search for an optimal method for cryopreservation of human epididymal sperm. METHODS: We collected semen samples from 76 men with obstructive azoospermia by percutaneous epididymal sperm aspiration and divided each sample into two parts to be cryopreserved with a self-made metal freezing plate (the experimental group) or by slow freezing (the control group), respectively. We measured the percentage of progressively motile sperm (PMS) with the computer-assisted semen analysis system and compared the membrane function, DNA fragmentation index (DFI), acrosin activity and morphological abnormality of the sperm between the two groups before and after cryopreservation. RESULTS: After thawing, both the percentages of PMS and hypotonically swollen sperm were significantly higher in the experimental than in the control group (ï¼»12.0 ± 7.5ï¼½% vs ï¼»8.0 ± 5.1ï¼½%, P < 0.05; ï¼»22.0 ± 17.5ï¼½% vs ï¼»18.0 ± 20.5ï¼½%, P < 0.05), though both decreased in comparison with the pre-freezing parameters (ï¼»20.7 ± 8.8ï¼½% and ï¼»30.0 ± 13.5ï¼½%) (P < 0.05). The sperm acrosin activity was remarkably higher in the experimental than in the control group after thawing (ï¼»75.2 ± 9.5ï¼½ vs ï¼»55.7 ± 8.3ï¼½ µIU/106sperm, P < 0.05), though decreased as compared with the baseline (ï¼»120.0 ± 10.5ï¼½ µIU/106 sperm, P < 0.05). No statistically significant differences were observed between the experimental and the control groups after thawing in the percentage of morphologically abnormal sperm (ï¼»98.7 ± 8.8ï¼½% vs ï¼»98.5±9.2ï¼½%, P > 0.05) or sperm DFI ï¼»38.2 ± 8.5ï¼½% vs ï¼»39.5 ± 10.2ï¼½%, P > 0.05), though both markedly elevated in comparison with the pre-freezing parameters (ï¼»97.2 ± 9.5ï¼½% and ï¼»30.8 ± 9.7ï¼½%) (P < 0.05). The post-thaw recovery rate of sperm was significantly higher in the experimental than in the control group (ï¼»65.2 ± 12.0ï¼½% vs ï¼»52.3 ± 18.0ï¼½%, P < 0.05). CONCLUSIONS: The self-made metal freezing plate, with its advantages of low cost, high efficiency, and easy operation, can be used as an effective method for cryopreservation of human sperm to achieve a high post-thaw sperm recovery rate, progressive sperm motility, and sperm acrosin activity.

An adapted carrier for the cryopreservation of human testicular spermatozoa.

RESEARCH QUESTION: Does the adapted carrier Cryoplus improve the quality of cryopreserved spermatozoa compared with the use of conventional containers, and what is the effect of the adapted carrier on clinical outcomes? DESIGN: Semen samples from 27 cases of oligozoospermia were used to investigate whether the adapted carrier improved cryopreserved sperm quality compared with the use of 0.25-ml straws and 2-ml cryogenic vials. Thirty testicular sperm samples were used to study the quality of testicular spermatozoa cryopreserved in the adapted carrier. The retrospective study included a further 104 men with azoospermia to investigate the clinical outcomes of testicular spermatozoa cryopreserved with the adapted carriers. Men with mostly obstructive azoospermia were included in this study. RESULTS: The adapted carrier improved cryopreserved spermatozoa motility of semen samples compared with 2-ml cryogenic vials but not compared with 0.25-ml straws. No differences were found in cryopreserved sperm DNA fragmentation among the three carriers. Fertilization and good-quality embryo rates were similar in ICSI cycles using fresh or cryopreserved testicular spermatozoa. Additionally, no difference was evident between frozen-thawed embryo transfer cycles using fresh or cryopreserved testicular spermatozoa in clinical pregnancy, implantation, miscarriage, live birth rates or birth weight. CONCLUSIONS: The adapted carrier improved the cryopreserved sperm motility compared with the effects of 2-ml cryogenic vials. The outcomes of intracytoplasmic sperm injection and frozen-thawed embryo transfer outcomes indicate that testicular spermatozoa cryopreserved using the adapted carrier is not inferior to fresh testicular spermatozoa. The use of the adapted carrier for cryopreserving human testicular spermatozoa especially from obstructive azoospermia is simple and effective.

Effects of two freezing methods and two cryopreservation media on post-thaw quality of stallion spermatozoa.

Glycerol-based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post-thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol-based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio® ). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.

Electric ultrafreezer (- 150 °C) as an alternative for zebrafish sperm cryopreservation and storage.

Zebrafish sperm cryopreservation is a fundamental methodology to manage and back-up valuable genetic resources like transgenic and mutant strains. Cryopreservation usually requires liquid nitrogen for storage, which is expensive and hazardous. Our objective was to evaluate if electric ultrafreezers (- 150 °C) are a viable alternative for zebrafish sperm storage. Zebrafish sperm was cryopreserved in the same conditions (- 20 °C/min), stored either in liquid nitrogen or in an ultrafreezer, and thawed after 1 week, 1 month, and 3 months. Sperm motility, membrane integrity, and fertilization ability were assessed. There were no significant differences in motility and hatching rate throughout storage time. Additionally, we aimed at understanding if cryopreservation directly in an ultrafreezer (- 66 °C/min) could improve post-thaw sperm quality. Freezing at - 20 °C/min was performed as before, and compared to samples cryopreserved with a fast cooling rate by placing directly in an ultrafreezer (- 66 °C/min). Sperm quality was assessed according to motility, viability, DNA fragmentation, and apoptosis (annexin V). The - 66 °C/min cooling rate showed significantly higher membrane and DNA integrity, and lower number of cells in late apoptosis in comparison to the other treatments. This study showed that zebrafish sperm cryopreservation and storage in an ultrafreezer system is possible and a fast cooling rate directly in ultrafreezer improves post-thaw sperm quality.

Gel-coated tubes extend above-freezing storage of honey bee (Apis mellifera) semen to 439 days with production of fertilised offspring.

Honey bees are an important agricultural species; however, relatively little work has been done to improve artificial reproductive technologies for this animal. The collection and distribution of germplasm for breeding and conservation is critical for improving managed honey bee populations and conserving threatened subspecies. The most efficient method of controlling breeding in honey bees is by artificial insemination. The collection of semen for insemination requires the use of antibiotics, which is especially critical if semen is to be stored for any length of time. The introduction of antibiotics is normally done through a balanced salt solution. In this study we compare, at two temperatures, the storage of undiluted semen in antibiotic-gel-coated capillary tubes with storage of semen diluted in a balanced salt solution containing antibiotics. Live-dead cell staining and artificial insemination of honey bee queens were performed at 45, 99 and 439 days after collection of the semen. In every case the antibiotic-gel-coated tube storage method at 14°C produced a higher percentage of fertilised offspring. This study demonstrates the longest period of time spermatozoa have been stored above freezing while maintaining fertilisation capacity.

Porcine sperm vitrification I: cryoloops method.

The aims of this study were to evaluate porcine sperm vitrification in cryoloops, with and without two different cryoprotectants and assess two warming procedures. Extended (n = 3; r = 4) and raw (n = 5; r = 2) semen was diluted in media without and with cryoprotectants (4% dimethylformamide and 4% glycerol) to a final concentration of 20 × 106 spermatozoa ml-1 and vitrified using the cryoloops method. Two warming procedures were evaluated: rapid method (30 s at 37°C) and an ultra-rapid method (7 s at 75°C, followed by 30 s at 37°C). Total motility (phase contrast), sperm viability (6-carboxifluorescein diacetate and propidium iodide stain), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated before and after vitrification and analysed using Friedman's test. In all media, the only seminal parameters that were maintained after vitrification were chromatin condensation and integrity. Vitrification of porcine spermatozoon using cryoloops, both in the presence or absence of cryoprotectants and independent of the warming procedure used, permits conservation of sperm chromatin condensation and integrity. It would be interesting to further verify this by producing porcine embryos using vitrified spermatozoon with intracytoplasmic sperm injection.

Improvement of common carp (Cyprinus carpio) sperm cryopreservation using a programable freezer.

The applicability of a programmable freezer for the increased-scale cryopreservation of common carp sperm was investigated. The effect of different equilibration times, cryopreservation methods, extenders, dilution ratios, activating solutions on the post-thaw motility of common carp sperm was investigated. The suitable post-thaw storage time-interval as well as fertilizing capacity of cryopreserved sperm was also examined. The motility, curvilinear velocity (VCL) and straightness (STR) values did not decrease significantly during 60min of equilibration neither in equilibrated nor thawed groups. Motility parameters of thawed sperm were similar using a conventional cryopreservation technique using a polystyrene box [motility (33%), VCL (47µm/s) and STR (88%)] and a programmable freezer: [motility (32%), VCL (54µm/s) and STR (89%)]. The highest motility and VCL was measured with a sugar based extender (grayling extender) at a ratio 1:9 (motility: 52%, VCL: 76µm/s) and 1:20 (motility: 49%, VCL: 76µm/s). The activating solution for cyprinids (ASC) could prolong sperm movement up for 2min. A storage time of six hours following thawing did not have a significant effect on the motility parameters of thawed carp sperm. Agglutination was observed during cryopreservation of an elevated volume of sperm whereas motility 47%, VCL 62µm/s and STR 91% were measured after thawing. Fertilization rate with thawed sperm (32%) was significantly lower compared to the control group (73%). According to our results, the developed method using a programmable freezer is suitable for the cryopreservation of elevated number of straws. However, carp sperm agglutination during freezing may have a negative effect on the fertilizing capacity.

Standardisation of a novel sperm banking kit - NextGen(®) - to preserve sperm parameters during shipment.

Many male patients diagnosed with cancer are within their reproductive years. These men are advised to freeze their spermatozoa prior to the start of cancer treatment. Very often, sperm banking facilities may not be readily available and patients may be required to travel to distant sperm bank centres. Our objective was to design and standardise a remote home shipping sperm kit that allows patients to collect a semen sample at home and ship it overnight to a sperm bank. A total of 21 semen samples and two transport media (refrigeration media and human tubal fluid) and five different combinations of ice packs were tested for maintaining desired shipping temperature. Ten semen samples were assessed for pre- and post-shipment changes in sperm motility, membrane integrity, total motile spermatozoa and recovery of motile spermatozoa. Even though motility, membrane integrity and total motile spermatozoa declined both in samples examined under simulated shipped conditions and in overnight-shipped samples, the observed motility and total motile spermatozoa were adequate for use with assisted reproductive techniques. Using refrigeration media, cooling sleeve and ice packs, adequate sperm motility can be maintained utilising NextGen(®) kit and these spermatozoa can be used for procreation utilising ART techniques such as intracytoplasmic sperm injection.

Spermiotoxicity of commercial condoms made from polyurethane, polyisoprene and latex, using domestic ruminants as an experimental animal model.

The use of condoms could provide a means of collecting high-quality spermatozoa from different species under physiological ejaculation conditions. However, certain condom materials may affect sperm functionality. This study examined the spermiotoxicity of different commercial condom materials towards ram and goat spermatozoa. Sperm samples were diluted in Tyrode's medium and placed in contact with a piece of condom material (polyurethane, polyisoprene or latex) and incubated for 30 or 90 min. Contact time in the polyisoprene and latex treatments affected some sperm variables; no such effects were seen, however, in the polyurethane treatments. For ram spermatozoa in contact with polyisoprene, the percentage of dead spermatozoa with a damaged acrosome increased at 90 min, while for spermatozoa in contact with latex, the percentage of live spermatozoa with an intact acrosome decreased. For goat spermatozoa in contact with both polyisoprene and latex, the percentage of dead spermatozoa with a damaged acrosome increased at 90 min, while for spermatozoa in contact with polyisoprene, the percentage of live spermatozoa with an intact acrosome decreased. In conclusion, latex and polyisoprene contain components that affect sperm motility, plasma membrane integrity and acrosome function. Polyurethane does not seem to reduce the quality of semen.

Effect of melatonin supplementation on cryopreserved sperm quality in mouse.

BACKGROUND: Antioxidants protect spermatozoa against cell damage during cryopreservation. OBJECTIVE: To investigate whether melatonin supplement in the extender may improve the quality of cryopreserved mouse sperm. METHODS: Kunming mice sperm frozen in extender R18S3 (18% (w/v) raffinose and 3% (w/v) skim milk) supplemented with melatonin were thawed and evaluated. RESULTS: Mouse spermatozoa were cryopreserved in the freezing extender R18S3 that contained melatonin at 0, 0.125, 0.25 and 0.5 mg/mL melatonin. The extender without melatonin supplement was associated with increased formation of reactive oxygen species (ROS) and decreased sperm motility. Melatonin supplement at 0.125 mg/mL significantly increased the progressive motility of sperm in comparison to other melatonin concentration or control. The percentage of thawed viable sperm with ROS was lower in the melatonin-treated groups than in untreated group. Melatonin supplement also increased antiapoptotic gene Bcl-xl expression in the thawed sperm. CONCLUSION: Supplement of 0.125 mg/mL melatonin could reduce oxidative damage and apoptosis.

Semen decontamination for the elimination of seminal HIV-1.

The risk of human immunodeficiency virus (HIV) transmission to the female partner, or potential offspring of an HIV-1 infected man can be reduced using semen decontamination procedures before assisted reproductive treatment (ART). The objective of this study was to determine the efficiency of decontaminating semen samples (n = 186) from 95 HIV-1 sero-positive patients. Aliquots of neat semen were submitted for viral validation by qualitative and quantitative polymerase chain reaction. Semen samples were processed by density gradient centrifugation in combination with a ProInsert™ tube after which aliquots of the processed sperm samples were analysed for the presence of HIV-1. Fifty-four percent of all tested neat semen samples tested positive for HIV-1 DNA, RNA or both (13.4%, 11.3% and 29.0%, respectively). From a total of 103 processed sperm samples that were submitted for viral validation, two samples tested positive for HIV-1 DNA and none for RNA. In conclusion, semen processing with the ProInsert™ followed by viral validation of processed sperm samples should be carried out when providing ART to couples where the male partner is HIV-1 sero-positive.

A new approach for cryoprotectant-free freezing of goat epididymal spermatozoa.

A cryoprotectant-free method was successfully used for rapid freezing of goat epididymal spermatozoa. Lowering sperm volume may increase the temperature exchange rate and improve the freezing output of spermatozoa. The aim of this study was to compare two different packaging types [0.25 ml French straws (FS) and 96-well immune plate (WIP)] for rapid freezing of goat epididymal spermatozoa. Eleven pairs of the goat testes were transferred to the laboratory; cauda epididymidides were dissected and sliced in TRIS-BSA solution for 15 min and temperature 33-35 °C. Sperm concentration was adjusted to 20 × 10(6) ml(-1), and the suspension was subjected to rapid freezing within FS or WIP. The volume of spermatozoa in WIP method was set at 25 µl. Sperm motility, viability and abnormalities, and sperm DNA integrity were compared between two devices. The results showed similar effectiveness of WIP and FS on post-thaw sperm parameters. In conclusion, for cryoprotectant-free rapid freezing of goat epididymal spermatozoa, it is recommended to use WIP instead of French 0.25 ml straws.

The Effect of Sperm Concentration and Storage Vessel on Quercetin-Supplemented Rabbit Semen During Chilled Storage.

Extending the shelf life of chilled rabbit spermatozoa is vital for the expansion of the farmed rabbit industry. This study evaluated the relationship between sperm concentration and packaging on in vitro quality of chilled rabbit semen over 96 h. Semen was collected from adult bucks (n = 4) and pooled at 37°C following evaluation. Pooled ejaculates were diluted with a Tris-based extender supplemented with 100 µm quercetin to a concentration of 15, 30 or 60 × 10(6)  spermatozoa/ml, packaged into plastic tubes or 0.5-ml straws and stored at 15°C. Sperm quality was assessed by computer-assisted sperm Analysis [total motility (tMOT)] and flow cytometry [viability, acrosome integrity, H2 O2 production, plasma membrane disorder, apoptosis and DNA fragmentation index (DFI)] at 0, 48, 72 and 96 h. From 48 h, concentrations of 30 and 60 × 10(6)  spermatozoa/ml reported the highest tMOT, irrespective of storage vessel (p < 0.05). Storage in straws reduced oxidative stress and improved plasma membrane stability. The %DFI, mean DFI and SD-DFI were increased in spermatozoa stored in tubes compared with straws (p < 0.05). Although the use of low sperm concentrations in artificial insemination doses would facilitate greater dispersion of genetically superior rabbit bucks, dilution to 15 × 10(6)  spermatozoa/ml had a detrimental impact on motility. As such, chilled storage at 30 × 10(6)  spermatozoa/ml may provide a suitable balance between motility and H2 O2 production to best maintain overall sperm function and should be evaluated in a large-scale AI trial.

DETERMINATION OF HEAT TRANSFER COEFFICIENTS FOR FRENCH PLASTIC SEMEN STRAW SUSPENDED IN STATIC NITROGEN VAPOR OVER LIQUID NITROGEN.

BACKGROUND: The use of mathematical models describing heat transfer during the freezing process is useful for the improvement of cryopreservation protocols. A widespread practice for cryopreservation of spermatozoa of domestic animal species consists of suspending plastic straws in nitrogen vapor before plunging into liquid nitrogen. Knowledge of surface heat transfer coefficient (h) is mandatory for computational modelling; however, h values for nitrogen vapor are not available. OBJECTIVE: In the present study, surface heat transfer coefficients for plastic French straws immersed in nitrogen vapor over liquid nitrogen was determined; vertical and horizontal positions were considered. MATERIALS AND METHODS: Heat transfer coefficients were determined from the measurement of time-temperature curves and from numerical solution of heat transfer partial differential equation under transient conditions using finite elements. The h values experimentally obtained for horizontal and vertically placed straws were compared to those calculated using correlations based on the Nusselt number for natural convection. RESULTS: For horizontal straws the average obtained value was h=12.5 ± 1.2 W m(2) K and in the case of vertical straws h=16 ± 2.48 W m(2) K. The numerical simulation validated against experimental measurements, combined with accurate h values provides a reliable tool for the prediction of freezing curves of semen-filled straws immersed in nitrogen vapor. CONCLUSION: The present study contributes to the understanding of the cryopreservation techniques for sperm freezing based on engineering concepts, improving the cooling protocols and the manipulation of the straws.

Comparison of Cryopreserved Human Sperm between Ultra Rapid Freezing and Slow Programmable Freezing: Effect on Motility, Morphology and DNA Integrity.

BACKGROUND: Cryopreservation of sperm is common methods to preserve male fertility. Sperm freezing, suggest slow programmable freezing caused lower change of sperm morphology than sperm freezing in vapor of liquid nitrogen. Ultra rapid freezing is easy to be worked on, less time, low cost and does not need high experience. OBJECTIVE: To compare the effect on sperm motility, morphology and DNA integrity of post-thawed sperm after ultra rapid freezing and slow programmable freezing methods. MATERIAL AND METHOD: Experimental study at laboratory of infertility unit, Department of Obstetrics and Gynecology, Faculty of Medicine Ramathibodi Hospital. Thirty-seven semen samples with normal semen analysis according to World Health Organization (WHO) 1999 [normal sperm volume ( 2 ml) and normal sperm concentration (≥ 20 x10(6)/ml) and sperm motility (≥ 50%)]. Semen samples were washed. Then each semen sample was divided into six cryovials. Two cryovials, 0.5 ml each, were cryopreserved by slow programmable freezing. Four 0.25 ml containing cryovials, were cryopreserved by ultra rapidfreezing method. After cryopreservationfor 1 month, thawedprocess was carried out at room temperature. Main outcomes are sperm motility was determined by Computer-Assisted Semen Analysis (CASA), sperm morphology was determined by eosin-methylene blue staining and sperm DNA integrity was assessed by TUNEL assay. RESULTS: Sperm motility was reduced significantly by both methods, from 70.4 (9.0)% to 29.1 (12.3)% in slowprogrammable freezing and to 19.7 (9.8)% in ultra rapid freezing (p < 0.05). Sperm motility decreased significantly more by ultra rapid freezing (p < 0.001). The percentage of normal sperm morphology and DNA integrity were also reduced significantly by both methods. However, no significant difference between the two methods was found (p > 0.05). CONCLUSION: Cryopreservation of human sperm for 1 month significantly decreased sperm motility, morphology and DNA integrity in both methods. However sperm motility was decreased more by ultra rapid freezing.