Easy and quick (EQ) sperm freezing method for urgent preservation of mouse strains.

Cryopreservation of mouse spermatozoa is widely used for the efficient preservation and safe transport of valuable mouse strains. However, the current cryopreservation method requires special containers (plastic straws), undefined chemicals (e.g., skim milk), liquid nitrogen, and expertise when handling sperm suspensions. Here, we report an easy and quick (EQ) sperm freezing method. The main procedure consists of only one step: dissecting a single cauda epididymis in a microtube containing 20% raffinose solution, which is then stored in a -80 °C freezer. The frozen-thawed spermatozoa retain practical fertilization rates after 1 (51%) or even 3 months (25%) with the C57BL/6 J strain, the most sensitive strain for sperm freezing. More than half of the embryos thus obtained developed into offspring after embryo transfer. Importantly, spermatozoa stored at -80 °C can be transferred into liquid nitrogen for indefinite storage. As far as we know, our EQ method is the easiest and quickest method for mouse sperm freezing and should be applicable in all laboratories without expertise in sperm cryopreservation. This technique can help avoid the loss of irreplaceable strains because of closure of animal rooms in emergency situations such as unexpected microbiological contamination or social emergencies such as the COVID-19 threat.

The Antibacterial and Antioxidant Roles of Buckwheat Honey (BH) in Liquid Preservation of Boar Semen.

In the present study, we hypothesized that buckwheat honey (BH) should be regarded as a potential alternative to antibacterial and antioxidant agent in liquid storage of boar semen. To this end, boar semen was firstly studied for in vitro dose tolerability to BH by measuring sperm progressive motility. The optimum progressive motility of boar spermatozoa was observed in extender with 0.5% and 0.6% BH addition. Afterward, sperm quality parameters, bacterial profile and composition, total antioxidant (T-AOC), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels of control, BH supplementation, antibiotics supplementation, and incorporated supplementation were compared during liquid storage period, to further investigate antibacterial and antioxidant properties of BH. The results showed that BH supplementation significantly improved sperm motility, acrosome integrity, plasma membrane integrity, inhibited opportunistic bacterial growth, and altered microbial compositions at the end of preservation. Additionally, T-AOC, SOD, and CAT levels were significantly higher in the BH supplementation group than those in the control and antibiotic supplementation group, whereas MDA level exhibited opposite change pattern. Importantly, BH addition to the extender was able to exert a synergistic effect in combination of antibiotic use. Our findings suggested that the appropriate concentrations (0.5% and 0.6%) of BH were added to the extender could act antibacterial and antioxidant roles in liquid preservation of boar semen.

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.

Cryopreservation of dog semen in a Tris extender with two different 1% soybean preparations compared with a Tris egg yolk extender.

Egg yolk is widely used as a cryoprotectant in dog semen extenders, but there is a risk of contamination with animal pathogens. In addition, egg yolk may vary in composition, making it difficult to standardize the extender. Lecithin is an animal protein-free alternative to egg yolk for semen cryopreservation. Recently, it was shown that 1% of soybean lecithin type II-S was better than 2% for freezing canine semen. The aim of the study was to compare two different types of soybean lecithin, with egg yolk as a control. Ejaculates from eight dogs were divided into three equal parts and diluted with a Tris-based extender, containing either 20% egg yolk, 1% soybean lecithin Type II-S or 1% soybean lecithin Type IV-S. The samples were then frozen. Sperm motility was evaluated by computer-assisted sperm analysis (CASA), acrosome integrity (FITC-PNA/PI) and sperm membrane integrity (SYBR-14/PI) post-thaw, as well as after 2 and 4 hr incubation at 37°C. Post-thaw sperm chromatin structure assay and plasma membrane integrity were evaluated by flow cytometry. Total motility, sperm plasma membrane integrity and acrosome integrity were significantly better in the egg yolk extender than in the two soybean lecithin-based extenders. Individual motility post-thaw differed more than in the fresh samples, illustrating individual differences in tolerance to the cryostress. The DNA Fragmentation Index (% DFI) was significantly lower in the Tris egg yolk (TEY) extender compared to any of the soybean-based extenders. The number of high green stained spermatozoa were significantly higher in Type IV-S compared to the control TEY extender. In conclusion, egg yolk was superior to the two lecithin-based extenders to cryopreserve canine semen.

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.

Cryopreservation of ferret (Mustela putorius furo) sperm collected by rectal massage and electroejaculation: Comparison of a decelerating and an accelerating freezing rate protocol.

The domestic ferret (Mustela putorius furo) provides a good model for developing new reproductive technologies for use with threatened related species. Such technologies could also be used in the reproductive management of this pet species. The present work reports an improved freezing protocol for ferret sperm. Semen was collected by electroejaculation plus rectal massage (in an attempt to reduce the electrical stimulation necessary) from five adult male ferrets, and then subjected to one of two freezing protocols: (a) from 5 to -35°C at 40°C/min, then from -35 to -65°C at 17°C/min, and finally from -65 to -85°C at 3°C/min-a decelerating freezing rate; and (b) from 5 to - 10°C at 5°C/min, and then from -10 to -130°C at 60°C/min-an accelerating freezing rate. After thawing, the viability and acrosomal integrity of the sperm frozen via the two-step accelerating method were better than those frozen via the three-step decelerating method (43.3 ± 3.5% and 71.2 ± 3.4% compared with 29.7 ± 3.7% and 58.8 ± 3.4% respectively; p < .05). No differences were seen between the methods with respect to sperm motility variables; most sperm (>90%) remained static with both freezing methods. In conclusion, although the method with accelerating freezing rate was associated with better post-thaw sperm viability and acrosome integrity values, neither of the two freezing methods tested provided adequate motility results after thawing. Combining rectal massage with electrical stimuli seemed to reduce the number of the latter required for successful sperm collection.

Novel micro-straw for freezing small quantities of human spermatozoa.

OBJECTIVE: To evaluate a novel micro-straw as an efficient, simple method for freezing a small number of human spermatozoa for intracytoplasmic sperm injection (ICSI). DESIGN: Prospective cohort study. SETTING: Sperm bank. PATIENT(S): Men with severe oligozoospermia or azoospermia undergoing a total of 143 ICSI cycles at the CITIC-Xiangya Hospital of Reproduction and Genetics from June 1, 2015, to June 31, 2019, and 20 donors at the Hunan Province Human Sperm Bank from 2001 to 2016. INTERVENTION(S): Analysis of sperm samples and clinical outcomes after sperm use. MAIN OUTCOME MEASURE(S): Clinical information, including number of motile sperm before and after freezing, freeze-thaw survival rates, two-pronuclear fertilization rates, clinical pregnancy, and early pregnancy loss rates after sperm use. RESULT(S): In the feasibility experiment using the micro-straw, we found a freeze-thaw survival rate of 73% ± 8.3% and no difference in normal sperm morphology, normal acrosome integrity, or DNA fragmentation index between the micro-straw and 1.8-mL cryotubes. The prospective cohort included 1,325 cases, and we collected sperm from testicular, epididymis, and ejaculation sources. We observed motile sperm in 1,294 (97.6%) of 1,325 frozen-thawed samples. Postthaw sperm were available for ICSI in 140 (97.9%) of 143 of cycles. The fertilization, cleavage, and high-quality embryo rates were 1,007 (81.7%) of 1,233; 995 (98.8%) of 1,007; and 537 (53.9%) of 995, respectively. Sixty-nine (49%) clinical pregnancies were achieved, and the miscarriage rate was 6 (8.6%) of 69. CONCLUSION(S): The micro-straw is suitable and clinically useful for the cryopreservation of small numbers of spermatozoa.

Modification of French mini-straw plug position for cryopreservation of small doses of bull sperm.

Greater than optimal diluting of semen for producing a large number of doses containing relatively small numbers of sperm can lead to compromised quality of sperm, post-thawing. In the present study the French mini-straw plug position was modified and the effect of re-positioning was evaluated on the quality of sperm after thawing subsequent to cryopreservation of small doses of sperm. Four types of mini-straws were used based on the position of cotton plug including no plug displacement (Type 1; Manufacturers location for plug-placement in 0.25 mL French mini-straws), and Type II, III, and IV with re-positioning the cotton plug being 2.5, 5, and 7.5 cm, respectively, further from the manufacturer's placement location. Each ejaculate was proportioned into four Aliquots (I, II, III, and IV) and diluted to 80, 60, 40, and 20, million sperm/mL, respectively. Aliquot I was placed in all types of straws, while Aliquots II, III, IV were placed only in Type I straws. Semen straws were equilibrated, cryopreserved and sperm kinetic and functional variables were evaluated post-thawing. The results indicate that in Aliquots III and IV there were lesser (P < 0.05) values for sperm kinetic and function variables compared with sperm from Aliquot I (i.e., unmodified mini-straw). In conclusion, cryopreservation of small doses of sperm in modified French mini-straws resulted in acceptable values for kinetic and function variables, post-thawing.

Effects of cashew gum and nanoparticles on cooled stallion semen.

BACKGROUND: Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes at 4 °C. RESULTS: Aliquots were analysed by computer-assisted sperm motility analysis on the day of collection, and after 24 h and 48 h of cold storage. After 48 h, the total motility with NP1 (78.53 + 6.31%) was similar to control 85.79 + 6.31% at 0 h. The same pattern was observed for progressive motility. Membrane integrity assessed by flow cytometer was similar between control, TOC and G1 at all storage times. The DNA fragmentation in the control group increased at all time points, whereas chromatin integrity was maintained after 24 h in TOC and NP0.5 compared to 0 h. There was no increase in the proportion of live spermatozoa producing hydrogen peroxide, but there was a tendency for an increased proportion of spermatozoa in the live superoxide category in CG1 after 24 h cooled storage. CONCLUSIONS: The addition of CG or CG-derived NP to extender for stallion semen was not harmful to the sperm cells.

Aseptic Technology for Cryoprotectant-Free Vitrification of Human Spermatozoa by Direct Dropping into Clean Liquid Air: Apoptosis, Necrosis, Motility, and Viability.

This study aimed to compare the quality of human spermatozoa vitrified by direct plunging into liquid nitrogen vs. liquid air. Spermatozoa were divided into three groups: fresh spermatozoa (Group F) were used as a control. Spermatozoa suspension (20 µl) was vitrified in open granules by direct dropping into liquid nitrogen (Group LN) or clean liquid air (Group LA). After warming at 37°C, the progressive motility rate of Group F was reduced from 65.9 ± 2.5% to 34.0 ± 1.9% (Group LN) and 38.1 ± 2.3% (Group LA), respectively (P1-2,3 < 0.05). The reductions in viability were 65.6 ± 2.2%, 29.0 ± 1.8%, and 36.6 ± 2.6% for Groups F, LN, and LA, respectively (P1-2,3 < 0.05). Comparing spermatozoa vitrified in liquid nitrogen vs. liquid air, no significant differences were detected in motility (34.0 ± 1.9% vs. 38.1 ± 2.3%), viability (29.0 ± 1.8% vs. 36.6 ± 2.6%), early apoptosis (13.8 ± 1.5% vs. 14.3 ± 1.8%), late apoptosis (45.5 ± 1.8% vs. 43.7 ± 2.2%), and necrosis (19.5 ± 2.0% vs. 15.0 ± 1.8%; p > 0.01 for all respective differences). There was a statistical tendency for increasing rates of "progressive motility" and "viability" and decreasing rates of "apoptosis" and "necrosis" when comparing spermatozoa vitrified in liquid air vs. liquid nitrogen. It is concluded that cryoprotectant-free vitrification by the direct dropping of human spermatozoa in a clean cooling agent (liquid air) is a good alternative to the use of nonsterile liquid nitrogen and can be used to cool cells while minimising the risk of microbial contamination.

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.

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.

[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.

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.

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.

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.

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.

Development of sea bream (Sparus aurata) semen vitrification protocols.

The long-term goal of this research project is to set up efficient protocol that can be used to develop a standardized approach for vitrification of marine fish spermatozoa. In particular, the aim of the present study was to develop a vitrification protocol for sea bream (Sparus aurata) spermatozoa. To draw up the protocol, we tested two different dilution media (1% NaCl and Mounib medium), three different vitrification devices (loops, drops and cut straws), different cryoprotectants (CPs) and three different equilibration times (30, 60 and 120 s). The effect of the different vitrification procedures on spermatozoa quality was checked by measuring spermatozoa motility rate and viability, mitochondrial membrane potential and the fertilizing ability of both fresh and post-thawed gametes. The best result was obtained by dropping directly into liquid nitrogen 20 µl of spermatozoa suspension (drop-wise method) diluted with Mounib buffer containing 10% Me2SO + 10% glycerol. The addition of a mixture of anti-freezing proteins, AFPI and AFPIII, to Mounib buffer significantly increases the spermatozoa quality following vitrification so confirming the usefulness of AFPs in improving the quality of gametes subjected to the vitrification process. The present study proves that vitrification offers an alternative to conventional sperm cryopreservation also in this species.

Vitrification of testicular tissue from prepubertal cats in cryotubes using different cryoprotectant associations.

Protocols for the cryopreservation of testicular tissue are not yet established. In cats, few studies have been conducted on testicular vitrification using different cryoprotectant associations (CPAs). Thus, the objective of this study was to compare the effect of different CPAs on the vitrification of testicular tissue from prepubertal cats in cryotubes. We used 10 pairs of testicles, with each pair divided into 8 fragments that were distributed into different experimental groups. Two of these fragments were allocated into the control group (CG) and the other six were distributed according to the CPAs to be tested (dimethyl sulfoxide (DMSO)/glycerol (GLY), ethylene glycol (EG)/GLY, or DMSO/EG). The cryoprotectants were used at a final concentration of 5.6 M. The fragments were subjected to vitrification in cryotubes and after 1 week, they were warmed and processed for histomorphologic assessment, quantification of nucleolar organizer regions (NORs), and determination of cell viability. The DMSO/EG and EG/GLY groups presented the greatest cell separation from the cell basement membrane and the highest degrees of retraction of the basal membrane. In these aspects, DMSO/GLY did not differ from the CG and both were significantly superior to the other groups. In terms of cell distinction, visibility of the nucleus, and nuclear condensation, all the vitrified groups had significantly lower values than the CG, while the DMSO/GLY and EG/GLY groups did not differ between themselves. Through the quantification of NORs, the potential for cell proliferation of the CG was found to have a mean of 3.80, while DMSO/GLY presented a mean of 3.60, and thus there was no significant difference between these two groups. The proliferation potentials of both groups were significantly superior to that of the DMSO/EG (mean: 2.07) and EG/GLY (mean: 1.98) groups. In the CG and DMSO/GLY group, 91.8% and 64.2% of cells, respectively, were found to be viable. The cell viabilities of both groups were significantly superior to those of DMSO/EG (52.5%) and EG/GLY (57.10%). Vitrification in cryotubes combined with the use of the DMSO/GLY association was effective in maintaining the histomorphology, cell proliferation potential, and cell viability of testicular tissue from prepubertal cats after cryopreservation.

Stallion Sperm Cryopreservation Using Various Permeating Agents: Interplay Between Concentration and Cooling Rate.

In this study, modeling and experimental approaches were used to investigate the interplay between cooling rate and protectant concentration for cryopreservation of stallion sperm. Glycerol (GLY), ethylene glycol (EG), dimethylformamide (DMF), propylene glycol (PG), and dimethyl sulfoxide (DMSO) were tested as cryoprotective agents (CPAs), using concentrations up to 1500 mM and cooling rates ranging from 5°C to 55°C min-1. Modeling of the extent of sperm dehydration during freezing was done using previously determined values of the sperm membrane permeability to water to predict optimal cooling rates for cryopreservation. Sperm cryosurvival was experimentally determined through flow cytometric assessments on membrane intactness and using computer-assisted analysis of motility. Sperm could withstand exposure to 1500 mM concentrations prefreeze for all CPAs tested. The overall highest cryosurvival rates were obtained with DMF, followed by GLY and EG, whereas the use of PG and DMSO resulted in poor cryosurvival rates. Cryosurvival with DMF increased with increasing concentration, reaching a plateau at 500 mM, whereas for GLY and EG, an optimum concentration between 250 and 500 mM resulted in maximal survival. An optimal cooling rate was only observed at low CPA concentrations, whereas at higher concentrations, cryosurvival rates were not affected by the cooling rate. In the case of DMF, survival remained relatively high in the investigated range of concentrations and cooling rates, whereas with GLY and EG, a much narrower combination of CPA concentration and cooling rate resulted in optimal cryosurvival. Sperm cryopreserved with DMF showed altered motility characteristics indicating hyperactivation, which was not observed with GLY and EG. Optimal cooling rates that were predicted from calculated dehydration curves did not match experimentally determined optimal cooling rates.