Liquid Nitrogen Vapor Research Articles

INNOVATIVE ULTRA-RAPID VITRIFICATION METHOD FOR FIVE OR FEWER TESTICULAR SPERMATOZOA FROM NON-OBSTRUCTIVE AZOOSPERMIC MEN AFTER MICROSURGICAL TESTICULAR SPERM EXTRACTION (MICRO-TESE)

Clinical outcomes of ICSI when using only five or fewer testicular spermatozoa after cryopreservation have been unsuccessful and are considered to be inferior to those using testicular fresh spermatozoa from Micro-TESE. A possible cause of these poor results has been the lack of a successful freezing technique. In these cases, repeated Micro-TESE and simultaneous oocyte pick up has been the only available treatment. We developed an innovative ultra-rapid vitrification method for five or fewer testicular spermatozoa from non-obstructive azoospermic men after Micro-TESE. This study included 113 non-obstructive azoospermic men using Micro-TESE, it was conducted retrospectively at St. Mother Clinic between 2011 and 2018. Each motile spermatozoon was carefully aspirated tail first into the pipette, put into a 2-μl microdroplet media of the vitrification medium near the tip of the Cryotop (Kitazato Corporation, Tokyo, Japan) submerged in liquid nitrogen vapor for 2 min and then immediately plunged into liquid nitrogen. The vitrified spermatozoa were warmed by dipping them into a droplet media. Successfully recovered motile sperm were selected and used for ICSI. Number of patients, transfer cycles and collected sperms were 113, 192 and 560. Mean age of patients and their wives were 32.0±3.7y and 28.4±5.8y. Clinical pregnancy rate, miscarriage rate, live birth rate and number of live offspring were 24.0% (46/192), 19.6% (9/46), 19.3% (37/192) and 37 (Male: Female = 17: 20). Sperm recovery rate and survival rate were 90.3% (506/560) and 70.4% (356/506). Fertilization rate and mean number of transferred embryos were 51.6% (99/192) and 1.73 (1-2). Mean gestational weeks and mean body weight at birth were 39.23±5.27w and 2852.31±314.28g. No congenital anomalies were observed in any of the babies. This method for cryopreserving five or fewer spermatozoa from non-obstructive azoospermic men showed a recovery rate above 90% and a survival rate of about 70% which is the highest data ever been reported. The clinical pregnancy rate was 24.0% and 37 healthy babies were born.

Linking in‐situ and ex‐situ populations of threatened amphibians through genome banking

AbstractGenetic variation of the endangered Puerto Rican crested toad (PRCT; Peltophryne lemur) has dwindled over time in both wild and captive populations, leading to long‐term sustainability issues for the recovery program. To address this challenge, we propose that PRCTs can be used as a model species to show how in‐situ and ex‐situ populations can be linked through sperm biobanking and gamete transfer, expanding genetic variability of both populations. Male toads (n = 10) in Guayanilla, Puerto Rico were administered human chorionic‐gonadotropin (hCG) and luteinizing‐hormone‐releasing‐hormone analog (GnRHa) to stimulate spermiation. Sperm was collected noninvasively from 9/10 males, cryopreserved in 10% trehalose with either 10% N,N‐dimethylformamide (DMFA) or 10% dimethyl sulfoxide (DMSO), and transported in liquid nitrogen vapor to the National Amphibian Genome Resource Bank at Mississippi State University. Ultrasonography was used to identify females (n = 3) with mature oocytes for GnRHa‐induced oviposition of eggs for in‐vitro fertilization (IVF). Post‐thaw sperm motility was 28% and 25% for sperm cryopreserved with DMFA or DMSO, respectively. Of the 9,672 eggs used for IVF, 4% (n = 306/6,981) were fertilized with frozen–thawed sperm, compared with 20% (n = 525/2691) fertilized with fresh sperm controls. Overall, 46 toadlets were produced from frozen–thawed sperm. After 4.5 months of headstarting, 14 juvenile toads produced from various genetic crosses using frozen–thawed sperm were released to new sites in the wild, introducing unique genetic representation and new founder lines. After 1.5 years, 24 adult toads produced using frozen–thawed sperm continue to thrive in the captive collection, and one of these males has now produced an F2 generation of offspring with 5,085 tadpoles released to the wild. This transformational study is the first to produce reproductively viable adult PRCTs using cryopreserved sperm from hormonally‐induced wild males and captive females, introducing innovative methods that link in‐situ and ex‐situ populations of endangered amphibians to revolutionize genetic management.

Heat transfer modeling of shrimp in tunnel type individual quick freezing system

AbstractCryogenic freezers are commonly used for quick freezing of high‐value food products at a lower temperature. These freezers offer improved overall product quality and reduced dehydration and drip losses. An accurate prediction of heat transfer during the food freezing process is useful in deciding proper production, design, handling procedure, and amount of cryogen required. In this study, computational fluid dynamics (CFD) was used for numerical modeling of heat transfer phenomena in a freezing system during individual quick freezing (IQF) of shrimp. The residence time of shrimp in the developed IQF tunnel freezer was determined using modified Plank's equation and verified by experimentations. Modeling was done in two zones of the freezer: (i) Precooling zone containing cold liquid nitrogen (LN2) vapors that were utilized for precooling of incoming shrimp and (ii) freezing zone where LN2 was sprayed on shrimp before exiting freezer. Simulations of heat transfer in shrimp were performed using the Ansys CFD software at seven different locations of the IQF tunnel freezer. Results revealed that the core temperature of shrimp decreased from 304.5 K to 263 K as it passes through the freezer. In general, as compared to other segments of shrimp, core temperature of shrimp was the highest while the top portion was minimal. Dynamics of velocity distribution, thermal profile, and spray modeling results of cryogen in the freezer supported simulation model of shrimp. Overall, numerical modeling results are consistent (≤5% variation) with experimental values.Practical ApplicationsThe cryogenic freezer is widely preferred to obtain a superior quality of frozen food products owing to minimal defects (cracking and break down), drip loss, and freezing time compared to traditional mechanical freezers. The inefficient utilization of cryogen and handling issues results in a higher cost of food products frozen with IQF technology. Thus, an insight into heat transfer phenomena in cryogenic freezers may lead to improvement in the design, efficient utilization of cryogen, reduction of freezing time, and improvement in overall product quality. In conclusion, these would aid in reducing the cost of the technology.

Use of dimethylformamide to cryopreserve alpaca semen previously incubated with collagenase.

The objective of this study was to evaluate the effect of collagenase and two final dimethylformamide (DMF) concentrations (4% and 7%) on alpaca frozen-thawed sperm quality. A total of 25 ejaculates from 5 alpaca were obtained using electroejaculation. Each individual ejaculate was evaluated and then diluted 4:1 in a solution of 1mg/ml collagenase in HEPES-TALP medium and incubated for 4min at 37°C. Subsequently, samples were diluted in TRIS-fructose-citric acid-egg yolk and cooled to 5°C. Then, each sample was divided in two aliquots and DMF at final concentration of 4% or 7% was added, equilibrated for 1hr at 5°C and frozen over liquid nitrogen vapours. A Kruskal-Wallis test was used to evaluate the sperm morphometry, and Completely Random Block designs were used to analyse sperm motility, viability, membrane function and acrosome status. After collagenase incubation, none of the samples showed thread formation, and sperm parameters were preserved. Non-progressive motile sperm were higher (p<.05) in equilibrated samples (4% DMF: 31.8±8.3% and 7% DMF: 36.3±11.8%) compared to raw (10.1±4.3%) and frozen-thawed semen (4% DMF: 9.7±1.8% and 7% DMF: 7.5±3.2%). Sperm membrane function, membrane integrity and intact acrosomes were higher (p<.05) in raw semen (40.1±12.2%, 94.6±3.2% and 91.3±8.1%) compared to frozen-thawed samples (4% DMF: 19.8±4.7%, 53.2±2.7%, 65.7±8.7% and 7% DMF: 20.4±4.5%, 54.1±1.4%, 64.6±9.1%). Length of the sperm head was lower in frozen-thawed samples, being statistically different with 4% DMF compared to pre-freezing samples. The ratio between acrosome and head areas was greater (p<.05) in frozen-thawed samples. Incubation of raw alpaca semen with collagenase decreased the thread formation without affecting sperm quality. Frozen of collagenase treated alpaca semen with 4% or 7% DMF did not preserve the sperm parameters in thawed samples.

Kualitas post-thawing semen domba Merino dalam bahan pengencer berbasis susu skim-kuning telur yang ditambah isolat crude protein Tirosine Kinase

This study was conducted to determine the effect of crude protein tyrosine kinase (PTK) isolates supplementation into skim milk-egg yolk based diluent to maintain the quality of Merino ram spermatozoa. Four ejaculates of two Merino rams were divided into two groups for the control group (P0): Merino ram semen was diluted in skimmed milk-egg yolk based diluent, and the treatment group (P1): Merino ram semen was diluted in skim milk-egg yolk based diluent contained crude PTK 1,597 mg/ml diluent. All diluted semen was equilibrated for 2 hours at 5 °C and filled into 0.25 mL French straws. The filled straws were placed on steel racks (Cooltop, Minitube) held in liquid nitrogen vapour for 10 minutes at –140 °C, immersed immediately in liquid nitrogen at –196 °C, and stored for 48 hours for later assessment. Post-thawed semen samples were evaluated for spermatozoa motility, viability, and morphological abnormality. The results showed that the spermatozoa motility of fresh semen of Merino ram was 82.5 ± 2.89, which was qualified for freezing. The post-thawing spermatozoa motility, viability, and morphological abnormalities of Merino ram in the P1 group were 34.11 ± 3.26%, 38.00 ± 3.00%, and 12.89 ± 4.54%, respectively. It were higher (p &lt;0.05) than the control group of 24.44 ± 2.9%, 26.67 ± 3.32%, and 21.11 ± 3.02%. It was concluded that the addition of crude PTK isolates of 1.597 mg/ml skim milk-egg yolk diluent improved the quality of post-thawed spermatozoa of Merino ram.

P–023 Innovative ultra-rapid vitrification method for five or fewer testicular spermatozoa from non-obstructive azoospermic men after microsurgical testicular sperm extraction (Micro-TESE)

Abstract Study question What technique can be used to successfully cryopreserve five or fewer testicular spermatozoa from non-obstructive azoospermic men? Summary answer This method for cryopreserving five or fewer spermatozoa from non-obstructive azoospermic men showed a recovery rate above 90% and a survival rate of about 70%. What is known already Clinical outcomes of ICSI when using only five or fewer testicular spermatozoa after cryopreservation have been unsuccessful and are considered to be inferior to those using testicular fresh spermatozoa from Micro-TESE. A possible cause of these poor results has been the lack of a successful freezing technique. In these cases, repeated Micro-TESE and simultaneous oocyte pick up has been the only available treatment. Study design, size, duration Evaluation of the efficiency of cryopreservation by modified permeable cryoprotectant-free vitrification method (HTF supplemented with 0.1M sucrose and 10% SPS) for five or fewer testicular spermatozoa from 113 non-obstructive azoospermic men using Micro-TESE was conducted retrospectively at St. Mother Clinic between 2011 and 2018. Participants/materials, setting, methods This study included 113 non-obstructive azoospermic men. Each motile spermatozoon was carefully aspirated tail first into the pipette, put into a 2-μl microdroplet media of the vitrification medium near the tip of the Cryotop (Kitazato Corporation, Tokyo, Japan) submerged in liquid nitrogen vapor for 2 min and then immediately plunged in liquid nitrogen. The vitrified spermatozoa were warmed by dipping them into a droplet media. Successfully recovered motile sperm were selected and used for ICSI. Main results and the role of chance Number of patients, transfer cycles and collected sperms were 113, 192 and 560. Mean age of patients and their wives were 32.0±3.7y and 28.4±5.8y. Clinical pregnancy rate, miscarriage rate, live birth rate and number of live offspring were 24.0% (46/192), 19.6% (9/46), 19.3% (37/192) and 37 (Male: Female = 17: 20). Sperm recovery rate and survival rate were 90.3% (506/560) and 70.4% (356/506). Fertilization rate and mean number of transferred embryos were 51.6% (99/192) and 1.73 (1–2). Mean gestational weeks and mean body weight at birth were 39.23±5.27w and 2852.31±314.28g. No congenital anomalies were observed in any of the babies. Limitations, reasons for caution The maximum number of spermatozoa to which this method can be applied successfully is about 10. When the number of aspirated spermatozoa is over 10, some of them change direction and reach the mineral oil, and once this happens, they cannot be expelled out of the pipette. Wider implications of the findings: This technique is very useful for the cryopreservation of very small numbers of testicular spermatozoa (fewer than 10) in order to avoid or reduce Micro-TESE interventions. Trial registration number N/A

Supplementation of Pandanus conoideus Oil in Cryopreservation Diluents for Maintaining the Semen Quality of Ongole Grade Bull

Antioxidants such as tocopherol, ß-carotene, and polyunsaturated fatty acids (PUFA) from red fruit oil of Papua may be used to protect frozen semen. The study is aimed to test the effect of red fruit oil supplementation on motility, viability, and recovery rate of frozen sperm of Ongole-grade bulls. Semen was collected twice a month from eight 4-5-year-old male Ongole grade using an artificial vagina, followed by macro- and microscopical evaluations. Collected semen was divided into four tubes and diluted with tris egg yolk diluents (TEY) as a control, TEY supplemented with 0.5% red fruit oil (RFO) (TEY RFO0.5), TEY supplemented with 1% RFO (TEY RFO1.0), and TEY supplemented with 1.5% RFO (TEY RFO1.5). The diluted semen was then packed into the straw and equilibrated for 2, 4, and 6 h prior to frozen on liquid nitrogen vapor for 10 minutes. The observed variables in this study were sperm motility, sperm viability, and morphology after equilibration, after thawing, and recovery rate. The experimental design is a completely randomized factorial design. The data were analyzed using ANOVA and were further tested using Duncan multiple range test. The results showed that the sperm motility of fresh semen was 81.10±1.42%. The percentage of sperm motility in TEY RFO1.5 treatment at 6 h equilibration was 60.00±1.06%, significantly higher compared to TEY RFO1.0 and TEY RFO0.5. The percentage of post-thawing sperm motility in TEY RFO1.5 treatment was 62.40±1.09%. The best post-thawing sperm viability in TEY RFO1.5 was 80.70±1.20%, significantly increase from the treatment of TEY RFO1.0 and TEY RFO0.5. The recovery rate (RR) for TEY RFO1.5 treatment had the best percentage at 76.94%. In conclusion, RFO supplementation in semen diluents for 2 h of equilibration resulted in the best motility and viability at 0 h of post thawing observation.

Assessment of the Dynamics of Temperature Changes in the Knee Joint Area in Response to Selected Cooling Agents in Thermographic Tests.

Although local cryotherapy (LC) is performed with various cooling agents (CAg) such as ice, water, and gasses, in clinical practice, it is mostly performed with cooling gasses. Presently, LC with cooling gasses is very popular but the inference about the thermal (stimulus) effect on the tissues is mainly based on research carried out using ice packs. The proposed objective of the study was to evaluate the dynamics of temperature changes in the knee joint area in response to a 3-min exposure to liquid nitrogen vapors (LNVs), cold air (CA) and ice bag (IB). The study group included 23 healthy volunteers with an average age of 26.67 ± 4.56. The exposed (ROIE) and contralateral (ROINE) areas of the knee joint after exposure to CAg were observed. Immediately after 3 min of LC, the ROIE temperature dropped by 10.11 ± 0.91 °C after LNV, 7.59 ± 0.14 °C after IB and 6.76 ± 1.3 °C after CA. Significant tissue cooling was maintained up to 15 min after LNV (p < 0.01), 10 min after IB (p < 0.05) and 5 min after CA (p < 0.05). LC causes significant temperature changes both in ROIE and ROINE. The greatest cooling potential was demonstrated for LNV and the lowest for CA.

The impact of temperature patterns during storage of Scots pine and Norway spruce seeds on their germination and fungal infection rates

Relevance of the study. One of the ways to maintain the genetic diversity of forest-forming species is to preserve highquality seed material ex situ. However, the relationship between the diversity of pathogenic mycobiota and the duration and methods of forest tree seed storage remains underexplored. The results of research into this problem can be used in forest seed production and forest phytopathology.Materials and methods. For our study we used seeds ofРinus sylvestris L. and Picea abies (L.) Karst. of the orthodox type, harvested in the period of 1996–2011 and stored under different temperatures: +20°С, +4°С, –18°С, and in liquid nitrogen vapor (–182°С) since 2011. Prior to their storage, seed samples were dried to a moisture content of 4.2–4.4% and hermetically packed. Seed germination was tested before and after three, five and eight years of storage, following GOST 13056.6-97 standards. The level of seed infection and the composition of pathogenic fungi were assessed.Results. After eight years of storage at +20°С, pine and spruce seed germination capacity decreased by 13–60%, depending on the year of harvesting. Seed storage at –18°С and –182°С allowed us to prevent seed infection and preserve seed viability. In most cases, the germination energy and germination capacity were negatively correlated with the level of seed infection. The diversity of pathogenic (mold) fungi on the surface of seeds was represented by ten genera; the most common were saprotrophs: Aspergillus P. Micheli, Penicillium Link, Rhizopus Ehrenb., Scopulariopsis Bainier.Conclusion. At the present stage of research, the success of seed storage at low and ultralow temperatures was demonstrated. We recommend cryopreservation for the long-term storage of improved and valuable seeds.

Thermo-Hydraulic Analysis of a Horizontal HTS ZFC Levitating Bearing Concerning Its Autonomy Safety Service Time

Electromagnetic heating of high-temperature superconductors (HTSs) and non-perfect thermal insulation of cryostats cause liquid nitrogen (LN2) vaporization and consumption. This paper presents results from the thermo-hydraulic analysis of the maximum safety service time of the prototype of a horizontal axis radial levitation bearing with zero-field cooled (ZFC) HTS bulks. It first studies the temperature sensitivity of yttrium barium copper oxide (YBCO) bulks to the LN2 level, particularly when partially immersed. This, when the main component of heat flux is according to the c-axis of the bulk crystal structure, that is the case of the studied bearing prototype and in most of all cryostats of maglev vehicles. Measurements used two platinum resistance thermometers leaning the top and side surfaces of a non-magnetized ZFC YBCO bulk. Correlations between the temperatures measured by the two platinum resistors are made to determine the average temperature of YBCO bulk. The thermal-time constant associated with a YBCO bulk losing contact with LN2 is also determined for two conditions: 1) initial ZFC and 2) initial field-cooled (FC) under the influence of typical magnetic field values generated by the studied bearing permanent magnet (PM) rotor. Results indicate that the YBCO temperature evolution is not notably affected by Joule losses from initial magnetization currents for this bearing. Due to continuous PM rings, the magnetic field perceived by the YBCO bulks does not change in the ideal case of spinning with no vibrations. Another experiment shows that the heating by induction from vibration dynamics does not contribute notably to LN2 consumption. Finally, the safety service time is verified from measured LN2 mass and level evolution in the HTS bearing's stator, due mainly to defective thermal insulation from the room temperature. The process consisted of calculating the times that different leveled YBCO bulks experiments reached the critical temperature and using the conclusions taken from the initial study about the temperature sensitivity of single bulks to the LN2 level. 3D finite element simulations reinforce our experimental results.

Fortification of Tris Extender with Mifepristone, Sericin and Taurine Improves Velocity and Kinematics of Fresh and Frozen-thawed Bovine Spermatozoa

Background: The evaluation of bull fertility essentially involves semen analysis and conception rates among inseminated females. The conventional evaluation is relatively imprecise, time consuming and subjected to individual variations, while computer assisted sperm analysis (CASA) is a quite faster, precise and objective tool to identify differences in sperm motility and velocity/kinematics attributes and avoids subjective errors. The present study was planned to evaluate and compare the CASA traits of fresh and frozen-thawed semen of Gir and Murrah bulls in Tris extender without and with different antioxidant additives by adopting Biovis CASA.Methods: The semen ejaculates with greater than 75% initial motility from 3 Gir and 3 Murrah bulls were split-diluted at 100 million sperm per ml using Tris-citrate-fructose-yolk-glycerol (TFYG) extender without (control) and with three antioxidant additives, viz., Mifepriston (10 µg/ml), Sericin (5 mg/ml) and Taurine (4 mg/ml), were filled in French mini straws and frozen in liquid nitrogen vapour using a programmable biofreezer. The freshly diluted and frozen-thawed semen samples were assessed for sperm motion characteristics, velocity/kinematics using Biovis CASA. Result: The mean percentages of total motile spermatozoa, irrespective of additives, in freshly diluted and frozen-thawed semen were 85.50±0.92 and 48.76±1.69 for Gir bulls and 85.03±0.72 and 52.61±1.46 for Murrah bulls, respectively. The mean total motile as well as rapid and slow progressive motile sperm percentage were significantly enhanced by fortification of TFYG extender with Mifepristone than in control extender, while values for Sericin and Taurine were intermediary and statistically similar to Mifepristone. The per cent decline in total motile sperm due to freezing-thawing was more or less same in both the breeds for all the extender additives, being lowest in Mifepristone supplemented extender in cattle (32.27 vs 48.81%) and buffalo (31.27 vs 43.14%) semen. The rapid and slow progressive motile sperm also followed the same pattern. The values of VAP and VCL were significantly (p less than 0.05) higher and VSL lower in Murrah than Gir breed at post-thaw stage. The overall mean linearity (%), straightness (%), beat-cross frequency (hz), lateral head displacement (µm), wobbling index (%), dancing velocity (µm2/s) and dancing mean (µm2/s) of sperm were higher in Murrahs than in Gir semen with significant (p less than 0.01) difference at post-thaw stage. The fortification of extender with Mifepristone improved all these traits at post-thaw stage compared to other additives and control TFYG extender. The per cent decline in these traits due to freezing stress was much lower in buffalo sperm than the cattle sperm particularly with Mifepristone fortification. It was concluded that freezing-thawing stress adversely affected the motion and kinematics of both cattle and buffalo sperm and that Tris extender fortified with antioxidant additives, particularly Mifepristone @ 10 µg/ml, protected sperm from adverse effect of dilution and cryopreservation with improved post-thaw sperm quality.

Effect of Bioxcell® and Triladyl® Extenders and Removal of Seminal Plasma on Equilibrated and Cryopreserved Semen from South African Unimproved Indigenous Bucks

The objectives of the study were to evaluate the effect of two extenders (Triladyl® and Bioxcell®) and the removal of seminal plasma on indigenous buck’s semen. Semen was collected from six indigenous bucks using an electro-ejaculator. Raw semen was pooled and randomly allocated into six groups as follows: (i) Raw non-washed, (ii) Raw washed, (iii) Triladyl®-washed, (iv) Triladyl®-non-washed, (v) Bioxcell®-washed and (vi) Bioxcell®-non-washed. Both the Triladyl® and Bioxcell® washed semen samples groups were diluted (1:4 v/v) with Phosphate Buffered Saline (PBS) then centrifuged at 1500x g for ten min and seminal plasma was removed. The groups were analysed for spermatozoa motility rates using Computer-Aided Sperm Analysis (CASA). The spermatozoa viability was assessed using Eosin-Nigrosin, acrosome integrity using Spermac, chromatin structure using Acridine Orange, and mitochondria using JC-1 staining solutions. Semen samples were diluted (1:4 v/v) as follows: Triladyl® (washed and non-washed) or Bioxcell® (washed and non-washed) and then equilibrated at 5°C for 2 hours. Equilibrated semen samples in 0.25 mL French straws were placed 5 cm above a Liquid Nitrogen (LN2) vapour for 10 min, and stored for one month. Frozen semen straws per treatment group were thawed at 37°C for 30 seconds. Significant differences among the mean values of semen parameters were determined by Tukey’s test using ANOVA, GLM procedure of SAS version 12.1 of 2010. The spermatozoa progressive motility rate in non-washed semen extended with Bioxcell® was significantly higher (89.6±7.5a) compared with that of non-washed Triladyl®, washed Bioxcell® and Triladyl® (P&lt;0.05). Live spermatozoa percentage in washed semen extended with Triladyl® extender was reduced (27.7±17.1) significantly compared with the other groups (P&lt;0.05). There was a lower percentage of spermatozoa with high mitochondrial membrane potential in non-washed and washed semen extended with Bioxcell® (39.5±23.2 and 37.9±28.6, respectively) compared with that of non-washed and washed semen extended with Triladyl® (P&gt;0.05). The spermatozoa progressive motility rate in non-washed semen extended with Bioxcell® (58.5±10.0) extender was significantly higher compared with that of the other groups (P&lt;0.05). There was a higher live and normal spermatozoa percentage in non-washed semen extended with Bioxcell® (45.7±21.2) compared with that of the other groups (P&lt;0.05). In conclusion, Washing of seminal plasma in semen extended with Triladyl® was not essential, as it lowered viability, progressive motility and chromatin membrane integrity prior and post-cryopreservation. However, Bioxcell® extender was found to be more suitable for preserving spermatozoa during equilibration and freezing/thawing process of non-washed and washed buck semen.

Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program

The aim of the present study was to optimize the conventional method of sperm freezing in liquid nitrogen (LN&lt;sub&gt;2&lt;/sub&gt;) vapour for successful cryopreservation of Wallachian ram sperm, the genetic resources of the Czech Republic. Sperm in straws were frozen using the conventional freezing method via a static exposure of sperm doses to LN&lt;sub&gt;2&lt;/sub&gt; vapour, or by four different modified freezing methods. Under modified freezing, straws were frozen by a discontinuous, time-dependent decremental change in the distance between the straws and the surface of LN&lt;sub&gt;2&lt;/sub&gt;. The viability of sperm was evaluated by flow cytometry after sperm equilibration, and immediately after thawing. Besides the observed inter-sire and daily variation, the obtained results suggest the methodological weakness of the conventional freezing method via the static exposure of sperm doses to LN&lt;sub&gt;2&lt;/sub&gt; vapour. With the use of the optimized freezing procedure, all parameters of thawed sperm were significantly (P &amp;lt; 0.05) improved in comparison with the conventional method: percentage of thawed sperm viability increased up to 48.3%, percentage of sperm with plasma membrane damage after thawing decreased to 6.58%, percentage of sperm with acrosome damage decreased to 24.4%, and percentage of sperm with deteriorated mitochondrial activity decreased to 6.28%. In conclusion, our results suggest that an optimized freezing procedure should be routinely used instead of the conventional method to cryopreserve Wallachian ram sperm.

Effects of selective extenders on buffalo semen quality at physical and molecular level

This study was designed to investigate the effects of selective extenders on buffalo semen quality at physical and molecular level for screening a suitable extender for cryopreservation of buffalo semen. Semen was collected from four indigenous buffalo bulls using artificial vagina method twice in a week. After initial evaluation, each semen sample was divided into three aliquots and diluted with three different extenders (Tris-egg-yolk extender, Soya-milk extender and Andromed extender). After initial quality assessment the semen was frozen with liquid nitrogen vapor with a programmable bio freezer and finally stored at –196ºC liquid nitrogen. Post thaw semen quality evaluation was performed after 24 h of storage. The expression of aldoketoreductase family 1 member B1 (AKR1B1) and A-kinase anchoring proteins (AKAP4) transcripts in fresh and three extenders groups (post thaw) of buffalo sperm were observed. The total, progressive, static and slow motility and hypoosmotic swelling (HOS) reactivity of pre-freeze and post thaw sperms were varied significantly among Andromed, Tris-egg-yolk and Soyamilk based extender. Similar variations were also observed for different kinetic parameters of pre-freeze and post thaw buffalo sperm. However, no significant variations were found in AKR1B1 and AKAP4 genes expression among Andromed, Tris-egg-yolk and Soya milk extender groups considering pre-freeze and post thaw sperms characteristics. It may be concluded that, Tris-egg-yolk semen extender might be used for cryopreservation of buffalo semen at efficiency level similar as commercially available semen extender like Andromed.

Ram and Goat Semen Immunosexed and Diluted in Powdered Coconut Water-Based Preservation Medium (ACP101/102c).

Background: Sperm sexing aims to separate sperm populations in carriers of the “X” or “Y” chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c.Materials, Methods &amp; Results: Ejaculates from 5 rams and 5 goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P &gt; 0.05), with the difference being observed only between T1 and T2, and T3 (P &lt; 0.05). Regarding the relative percentage and sperm morphology, no statistically significant differences were observed (P &gt; 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species.Discussion: The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species.

34 Cellular effects of antifreeze proteins type I and III in extender for sheep semen cryopreservation

Antifreeze proteins (AFP) have been included in extenders for sperm cryopreservation to prevent ice crystal formation. Thus, this study assessed the effects of supplementing semen extender with two concentrations of AFP types I and III on the quality of frozen–thawed ram sperm. The hypothesis is that various types and concentrations of AFP enhance cryopreservation of ram sperm. Semen was collected from 4 rams, pooled in 6 replicates, and allocated into 1 of 5 treatments: Control (CONT, without AFP); AFP type I [0.1 (AFPI-0.1) or 0.5 (AFPI-0.5) µg±mL−1]; or AFP type III [0.1 (AFPIII-0.1) or 0.5 (AFPIII-0.5) µg±mL−1]. Straws were placed on a metal wire net frame at 37°C and placed in a refrigerator for 2h to cool them to 5°C (−0.25°C/min). After 2h for stabilisation, straws were cooled in liquid nitrogen vapor (−15.3°C/min) and subsequently immersed (−196°C). After thawing, samples from each treatment were evaluated microscopically (sperm kinetics, plasma membrane integrity, capacitation, hypoosmotic test, acrosome status and mitochondrial activity, chromatin condensation, morphology, binding to egg perivitelline membrane, and lipoperoxidation quantification). The normal distribution of residuals was determined by Shapiro-Wilk test and homoscedasticity by Levene’s test. Normally distributed variables were analysed with one-way analysis of variance (ANOVA), followed by Tukey’s test. The non-normally distributed were analysed by Kruskal–Wallis and Dunn’s test. The repeated-measures ANOVA in general linear model (GLM) was used to effects of concentration for each AFP type in paired samples. The Greenhouse-Geisser test was applied when sphericity was not considered, followed by the Sidak test. Values of P&amp;lt;0.05 were considered significant. Treatments affected (P&amp;lt;0.05) kinetic parameters, plasma membrane integrity, and morphology. Linearity was greater in AFPI-0.1 (56.6±3.1%, mean±s.e.m.), AFPI-0.5 (56.9±2.2%), and AFPIII-0.5 (64.7±6.2%) than in CONT (36.8±3.0%). Straightness was greater in all AFP-supplemented extenders (overall mean, 78.6±2.8%) than in CONT (63.2±0.8%). Plasma membrane integrity was greater in AFPI-0.1 (49.1±4.6%) and AFPI-0.5 (36.6±7.3%) compared with CONT (13.0±4.4%). All AFP groups had a greater percentage of normal sperm (overall mean: 74.3±1.3%) than CONT (65.3±1.9%). There were no significant differences in percentage of sperm with functional membrane (overall mean: 16.1±3.3%), normal acrosome (11.5±4.5%), mitochondrial activity (24.5±6.5%), chromatin condensation (98.8±0.4%), perivitelline membrane binding rate (194.0±44.5 sperm/mm2), and lipoperoxidation (556.7±20.5 TBARS ng±mL−1). In conclusion, the use of AFP, predominantly type I, had potential as a cryoprotectant for ram sperm, increasing sperm cell protection, with no adverse effects on potential fertilization capacity and did not increase reactive oxygen species. This research was funded by FAPERJ, CNPq, and CAPES (Finance Code 001).

The effect of L-glutamine and trehalose on dog sperm cryopreservation

This study aimed to test different doses of L-glutamine and trehalose in the canine semen diluent while determining their protective effects on spermatological and biochemical indices of the thawed samples. Semen samples were collected from three fertile dogs using the digital manipulation method. The mixed ejaculates were divided into five portions at 37 °C and diluted with additives. Five study groups were formed with L-glutamine (10 and 20 mM), trehalose (25 and 50 mM), and no additives (control). After the dilution, the semen samples were cooled for 1.5 h at 5 °C and frozen (-110 to -120 °C) in liquid nitrogen vapor. Then, they were stored at -196 °C. For spermatological evaluations, samples were thawed at 38 °C for 30 s. L-glutamine (20 mM) was found to be significantly different (P &lt; 0.05) and led to higher percentages of motility, membrane integrity, and acrosome integrity compared to the control group. Considering the total oxidant status (TOS) assay, the lower values were determined in all the antioxidant groups compared to the control group (P &lt; 0.05). Supplementing the semen extender with L-glutamine showed a higher total antioxidant status (TAS) concentration compared to the control group (P &lt; 0.05). As a result of this study, a higher protective effect was found in all the spermatological evaluations after thawing the frozen semen samples, especially in the group containing L-glutamine (20 mM).

ANTIULCEROGENIC EFFECT OF CRYOPRESERVED PLACENTA EXTRACT AND THE EFFECT OF LOW TEMPERATURES ON THE DIGESTIVE TRACT INJURED BY DICLOFENAC SODIUM IN THE EXPERIMENT

Today, the ulcerogenic effect of nonsteroidal anti-inflammatory drugs is a key factor that significantly limits their clinical use and is a serious medical and social problem, as these drugs are among the most commonly used drugs – they are used annually by about 5­–7% of the world's population. The aim is to characterize the antiulcerogenic effect of cryopreserved placenta extract and its application against the background of low temperatures in the model of diclofenac sodium-induced ulcerogenesis in rats according to macroscopic studies of the proximal and distal digestive tract. The study was performed on 42 male rats weighing 200–220 g. Acute diclofenac sodium-induced gastrointestinal damage was replicated by a single intragastric administration of diclofenac sodium to rats at a dose of 50 mg/kg. Euthanasia of animals was performed after 24 hours. The condition of the mucous membrane of the digestive tract was assessed on a scale and calculated integrated indicators – ulcer index and antiulcer activity. Cryocell-cryoextract of placenta was administered to rats intramuscularly at a dose of 0.16 ml/kg body weight. Cryoirrigation was performed once by local injection of liquid nitrogen vapor (temperature – 120˚C) for 10 s. It was found that diclofenac sodium at a dose of 50 mg/kg led to erosive-ulcerative damage to the gastric mucosa in 100% of rats, and the ulcer index was 3.9. The most pronounced leveling of the ulcerogenic effect of diclofenac sodium was observed against the combined preventive use of placental cryoextract and low temperature effect – the ulcer index was 12.6 times lower than that of rats with diclofenac sodium-induced ulcerogenesis without correction. Macroscopic evaluation of the distal gastrointestinal tract showed that the introduction of diclofenac sodium led to a statistically significant (p &lt; 0.05) lesion of the mucous membrane of the small and large intestine in 42.9% of rats. According to the magnitude of antiulcer effect (%) in the model of diclofenac sodium-induced ulcerogenesis, the investigated prophylactic approaches for antiulcer activity have the following priority: action of low temperatures + cryoextract of placenta (96.7%) &gt; cryoextract of placenta (92.1%) ~ esomeprazole (88.2%) &gt; action of low temperatures (72.1%). No lesions of both the small and large intestine on the background of the introduction of placental cryoextract in the model of diclofenac sodium-associated ulcerogenesis were detected.

32 An approach to invitro oocyte maturation and fertilization in threatened Saharan Dorcas gazelle (Gazella dorcas osiris) using frozen-thawed epididymal sperm cells

The Dorcas gazelle (Gazella dorcas), in the family Bovidae, is one of the smallest species of the genus Gazella. Its population is decreasing and it is classified as vulnerable by the International Union of Conservation of Nature Red List of Threatened Species. The subspecies Saharan Dorcas gazelle (G. dorcas osiris), originating in the central-western Saharan and Sahel regions, is almost extinct in the wild because of over-hunting. Thereon, assisted reproductive techniques (ARTs) could help in its population management. Here, we performed invitro maturation (IVM) of oocytes recovered from an adult female of Saharan Dorcas gazelle (10 years old) to attempt IVF with frozen-thawed epididymal sperm cells from an adult male (9 years old) of the same subspecies, following protocols for ARTs in cattle. Epididymal sperm was obtained after postmortem examination by cannulation of vas deferens with commercial semen extender Gent A (Minitub). Motility features were assessed using a computer-assisted sperm analysis system (Proiser SL), and viability was evaluated using eosin/nigrosin staining. After collection, viability (88.3%), total (17.8%) and progressive (8.7%) motility were assessed. A total of 820.5×106 sperm cells was obtained and diluted (1:1) in a semen extender with permeable cryoprotectants (Gent B, Minitub). Then, the sample was refrigerated in straws (200×106 mL−1; 0.5mL) at 4°C for 1h and exposed to liquid nitrogen vapors (LN2) for 10min before being directly plunged into and kept on LN2. A total of 35 oocytes were recovered by slicing, 27 of which were selected for IVM (24h, 38.5°C, 5% CO2 in humidified air). Motile sperm were obtained by centrifugation of thawed semen (10min, 1000×g) by performing a density gradient (BoviPure, Nidacon). Post-thawing viability (72.9%) and total (14%) and progressive (4%) motility were evaluated just before IVF. After IVM, 17 oocytes were incubated for IVF for 20h with 1×106 thawed sperm cells mL−1 (although the first polar body extrusion was observed only in 5 of the 17 oocytes before IVF; 29.4%). Also, 7 oocytes selected for IVM were vitrified and kept for the genetic bank, and 3 were fixed. Thereafter, all oocytes that underwent IVF were incubated for invitro embryo culture (38.5°C, 5% CO2, 5% O2 in humidified air) to avoid discarding any potential zygote. Cleavage was assessed at 48h post-IVF. From those, only 2 oocytes successfully achieved IVF out of the 5 that showed first polar body extrusion (40%), although cleavage was asymmetric and embryos did not progress. The rest of oocytes (12) did not achieved IVM, IVF, or cleavage. Results suggest that optimal protocols and appropriate individualized conditions for vulnerable nondomestic ungulates are required for successful ARTs. Future efforts are needed to improve preservation strategies for the gametes/embryo conservation of endangered species. JG and MRC contributed equally to this work. JG is supported by AGAUR (2018 FI_B 00236) and MRC by FPU (Training for Academic Staff) (FPU15/06029).

Effect of alpha lipoic acid and trehalose on ram semen cryopreservation

Bu çalışmada, koç sperma sulandırıcısına farklı dozlardaki Alfa Lipoik asit (ALA) ve Trehalozun eklenerek dondurulup çözdürülmesinden sonra spermatozoon motilitesi, plazma membran ve akrozom bütünlüğü (PMAI), mitokondrial membran potansiyeli ve mitokondrial reaktif oksijen türleri üzerine etkilerinin araştırılması amaçlanmıştır. 10 adet Pırlak koçtan suni vajen kullanılarak alınan spermalar. üç eşit parçaya bölünerek Alfa lipoik asit (1 mM), Trehaloz (100 mM) içeren ve hiçbir katkı maddesi içermeyen (kontrol) sulandırıcılar ile 5x108/ml spermatozoa olacak şekilde sulandırıldı. Spermalar + 4 ° C'de yaklaşık 2 saat ekibrasyona bırakıldı, sıvı azot buharında (-120 C⁰) donduruldu, 37 C⁰’de 30 saniyede çözdürüldü. Çözdürülen sperma örneklerinde motilite, 37 C⁰'de ısıtma tablalı faz kontrast mikroskop ile subjektif olarak, PMAI, mitokondrial membran potansiyeli ve mitokondrial reaktif oksijen türleri (MitoSOX+) flow sitometre ile değerlendirildi. En düşük motilite kontrol grubunda tespit edilirken en yüksek plazma membran akrozom bütünlüğü Trehaloz ve ALA gruplarında belirlendi (P&amp;lt;0,05). En düşük Hmmp ve en yüksek Lmmp değeri kontrol grubunda tespit edilirken en düşük MitoSOX+ Trehaloz grubunda belirlendi (P&amp;lt;0,05). Sonuç olarak; Pırlak koçlarda sezon dışı permanın dondurulmasında ALA ve Tehalozun spermatolojik parametreleri iyileştirdiği belirlenmiştir.