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This study evaluated the effects of freezing extenders, cryoprotectants and their concentrations, presence of Orvus Es Paste and freezing rates for cryopreserving dromedary camel sperm. Semen (five males; 2 ejaculates/male) was frozen in one of the following extenders (Green Buffer ® or INRA96 ® ), cryoprotectants (3 and 6% glycerol or ethylene glycol), with or without Orvus Es Paste and freezing at two different heights (1 and 4 cm) above liquid nitrogen. Sperm motility recovery parameters were evaluated post-thaw (0 and 1 hr), vitality and acrosome integrity (0 hr). Green Buffer showed higher total motility recoveries (p < .001). Higher percentage of cryoprotectant improved both total and progressive motility at 0 hr (p < .001; p = .003) and 1 hr (p < .001; p = .005). Acrosome integrity at thawing increased in the presence of ethylene glycol (p < .001) and Orvus Es Paste (p = .001). Kinematics were affected by extender, cryoprotectant concentration and Orvus Es Paste at 0 and 1 hr, and type of cryoprotectant only influenced them at 0h. Our findings showed strong interactions among type of cryoprotectant and concentration, and extender and Orvus Es Paste. Generally, combining Green Buffer, 3%-6% ethylene glycol or 6% glycerol without Orvus Es Paste, regardless of the freezing rates, yielded the highest post-thaw parameters for camel sperm., (© 2021 Wiley-VCH GmbH.)

The purpose of this research was to assess whether the presence of seminal plasma (SP) can improve sperm quality of rabbit spermatozoa stored at 16°C for 72 h and moreover evaluate the cryoprotectant effects of glycerol, N-N-Dimethylformamide (DMF), and N-methyl-2-pyrrolidone (NMP). Semen samples were pooled and divided in eight fractions. Four of them were diluted with INRA (extender A), INRA with 6% glycerol (extender B), INRA with 6% DMF (extender C), or INRA with 6% NMP (extender D), respectively. The other four fractions were centrifuged, and the supernatant was removed in order to eliminate SP. Each sample was then resuspended with extender A, B, C, or D, respectively. All samples were stored at 16°C and analysed at 4, 24, 48, and 72 h by ISAS ® , vitality test, HOS test, and acrosome integrity test. After analyse of the results, SP samples showed a significantly higher percentage (P=0.020) in the HOS test (71.9 ± 1.6%) than non-SP samples (66.5 ± 1.6%). Non-SP samples had better results for kinematic parameters. Extenders A and C showed great results for the percentage of motile spermatozoa (63.1 ± 4.3% and 63.4 ± 3.7%, respectively), vitality (88.9 ± 2.6% and 87.7 ± 2.7%, respectively), and HOS test (68.9 ± 1.4% and 75.2 ± 1.4%, respectively). Extenders B and D showed worse data for sperm quality. These results suggest that SP has a protective effect on rabbit sperm membranes and maintains better sperm motility. The addition of glycerol and NMP to INRA does not improve rabbit sperm quality; nevertheless, the DMF cryoprotectant exerts a protective effect on the membrane of spermatozoa, improving seminal quality during rabbit sperm preservation at 16°C.

Freeze-drying (FD) technique has been applied as an alternative technology to preserve gene resources to allow simple sperm preservation and shipment at 4 °C. Nevertheless, DNA sperm might be damaged by mechanical or oxidative stress throughout FD procedure. Therefore, suitable protection to maintain DNA integrity is required. The aim of this study was to determine the effect of rosmarinic acid (RA) as an antioxidant and two chelating agents (EGTA and EDTA) on the DNA integrity of freeze-dried rabbit sperm after storage of the samples at 4 °C and room temperature for 8 months. Rabbit sperm were freeze-dried in basic medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with 50 mM EGTA (1), 50 mM EGTA plus 105 μM RA (2), 50 mM EDTA (3) or 50 mM EDTA plus 105 μM RA (4). Semen samples were kept at 4 °C and room temperature during 8 months. After rehydration, DNA integrity was evaluated with Sperm Chromatin Dispersion test observing that DNA fragmentation was higher when semen samples were freeze-dried with EGTA (10.9%) than with EDTA (4.1%) (p < 0.01). Furthermore, RA acted better under adverse conditions and no significant differences were found in temperature storage. Summarizing, FD is a method that can allow simple gene resources preservation among 4 °C to 25 °C during 8 months and transportation without the need for liquid nitrogen or dry ice. EDTA chelating agent is the most suitable media for freeze-dried rabbit sperm and the addition of RA protects the DNA against the oxidative stress caused during FD procedure., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The presence of DNA protective agents in the medium is necessary to maintain sperm functionality after freeze-drying procedure. The objective of this study was to investigate the effect of chelating agents, ethylene diaminetetraacetic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA), in combination with rosmarinic acid (RA) on DNA integrity of freeze-dried boar sperm. We also examined the effect of these agents on the in vitro developmental ability of porcine oocytes following sperm injection (ICSI). Heterospermic mix, obtained from ejaculated sperm of three boars, was freeze-dried in two different chelating agents' media: 50mM EDTA or 50mM EGTA, and in these media supplemented with 105μM of rosmarinic acid. Frozen-thawed sperm was used as control. After rehydration, samples were subjected to DNA damage detection using Sperm Chromatin Dispersion test. ICSI was performed to verify the ability of freeze-dried sperm to participate in embryonic development. Five replicated trials were carried out for each group. In the presence of rosmarinic acid, the percentage of spermatozoa with DNA damage decreased significantly (p=0.010), without differences between the two chelating agents combination. EDTA solution preserves more efficiently DNA integrity of boar sperm than EGTA solution (p=0.002). There were no significant differences among the studied groups related to the blastocyst formation rate. Results suggested that the addition of rosmarinic acid to the medium improves sperm DNA integrity after freeze-drying, but does not promote fertilization and blastocyst development. We also observed a similar percentage of embryos production with freeze-dried and with frozen-thawed sperm., (Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Freeze-drying (FD) has been proposed as an alternative method to preserve spermatozoa. During the FD procedure, sperm DNA might become damaged by both freezing and drying stresses caused by the endonucleases, the oxidative stress and the storage conditions. We examined the DNA integrity of dog sperm freeze-dried with two kinds of chelating agents in FD buffers and storage at two different temperatures. Ejaculated sperm from four dogs were suspended in basic medium (10 mM Tris-HCl buffer+50 mM NaCl) supplemented with 50 mM EGTA or with 50 mM EDTA and then freeze-dried. Sperm samples were stored at 4°C as room temperature, and the analysis of DNA damage was performed after a month and 5 months of storage using a Sperm Chromatin Dispersion test. We found four different sperm populations according to the size of the halos around the sperm head: (1) absent halo, (2) <6 μm, (3) 6-10 μm, (4) >10 μm. All of them coexisted in each freeze-dried dog semen samples and differed significantly among different treatments. The highest percentage of spermatozoa with halo >10 μm was obtained when the semen samples were freeze-dried in EDTA medium and stored at room temperature for five months. Results suggested that both, the kind of chelating agent as well as storage temperature and period, influenced DNA integrity of freeze-dried dog sperm., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Sugars are the energetic source for sperm to maintain the metabolic process, and the antibiotics slow down sperm degradation., Objective: To study the effects of rosemary honey as energy source and cryoprotectant in combination with garlic as a natural antibiotic on the quality of ram spermatozoa upon cooling., Materials and Methods: The ejaculates from three rams were evaluated at different times during cooling to determine its post-dilution quality., Results: Glycerol and dimethylformamide in conjunction with honey and garlic significantly improve the survival of spermatozoa., Conclusion: The addition of honey and garlic reduces sperm deterioration when stored at 4 degree C.

The aim of this study was to evaluate the effect of fennel and sage extracts and the influence of the egg yolk source (fresh or pasteurized) on the success of freezing boar epididymal spermatozoa. In experiment 1, epididymal sperm was recovered by flushing and cryopreserved in a lactose-egg yolk solution supplemented with various concentrations (10, 5 and 2.5 g/L) of sage or fennel. Sperm quality was evaluated (motility, viability, HOST and acrosome integrity) at 0 h and 2 h after thawing. Fennel 10 g/L and sage 5 g/L and control (no extracts) were selected for experiment 2 which also compared fresh or pasteurized egg yolk in the freezing extender and measured DNA integrity of the frozen sperm. Results showed that the interaction between fennel and sage antioxidants with fresh egg yolk significantly improved post thaw sperm quality and protected boar epididymal spermatozoa from cryopreservation damage as a result of oxidative stress.

During cryopreservation, oxidative stress exerts physical and chemical changes on sperm functionality. In the present study we investigated the antioxidant effect of rosmarinic acid (RA) on quality and fertilising ability of frozen-thawed boar spermatozoa. Ejaculates collected from mature boar were cryopreserved in lactose-egg yolk buffer supplemented with different concentrations of RA (0 μM, 26.25 μM, 52.5 μM and 105 μM). Motion parameters, acrosome and plasma membrane integrity, lipoperoxidation levels, DNA oxidative damage (8-hydroxy-2-deoxyguanosine base lesion) and in vitro fertilisation ability were evaluated. Total and progressive motility were significantly higher in experimental extenders with RA than in the control (P<0.05) at 0 and 120 min post-thawing. The plasma and acrosomal membrane integrity were improved by supplementation with 105 μMRA (P<0.05). Negative correlation between RA and malondialdehyde (MDA) concentration were determined (P<0.05). After thawing, the percentage of spermatozoa with oxidised DNA did not differ between extenders, however, at 120 and 240 min post-thawing, the samples supplemented with 105 μMRA showed the lowest DNA oxidation rate (P<0.05). The penetration rate was significantly higher on spermatozoa cryopreserved with 105 μMRA (P<0.05). The results suggest that RA provides a protection for boar spermatozoa against oxidative stress during cryopreservation by their antioxidant properties., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Freeze-drying spermatozoa is a developing technique that facilitates semen storage and transport. However, freeze-dried sperm exhibits impaired DNA integrity, which is associated with reduced fertilizing ability. Boar spermatozoa were freeze-dried in three different freeze-drying EDTA buffers with trehalose (75mM) and lactose (75mM) (EDTA-TL), (2) with sucrose (75mM) and lactose (75mM) (EDTA-SL) or just lactose (150mM) (EDTA-LL) using two freeze-drying protocols. In experiment 1 a one-step protocol was used and in experiment 2 a two-steps protocol was used. Spermatozoa were stored in1.5 mL cryo-tubes and 1.5 mL glass ampules at both 16 degree C and 25 degree C for 1 month. Successfully freeze-dried spermatozoa were stained with acridine-orange to assess chromatin stability. Freeze-drying was most successful when the 2-step protocol was used (experiment 2). Chromatin stability was greater in samples stored in glass ampules compared to cryo tubes. Chromatin stability was also greater in samples freeze-dried in EDTA-LL compared to EDTA-SL or EDTA-TL buffers. Spermatozoa freeze-dried in EDTA-LL and stored for 14 and 28 days at either 16 degree C or 25 degree C were utilized for ICSI. Two pronuclear formation wasgreatest using spermatozoa stored at 25 degree C (69.23%) and for 28 days (50%). Although 16 degree C spermatozoa samples had better stable chromatin, 25 degree C spermatozoa samples offered better two pronuclear formation results. In conclusion, boar spermatozoa freeze-dried using media containing disaccharides exhibit high chromatin stability and are able to fertilise oocytes following ICSI. Disaccharides may therefore advance the development of freeze-drying techniques for spermatozoa enabling ease of sperm storage and transportation.

The aim was to assess the in vitro effect of pasteurized egg (PE) and rosemary (Rosmarinus officinalis) on frozen-thawed ram semen. Ejaculates from three mature rams of the Rasa Aragonesa breed were cryopreserved using a 2-step dilution method (Fraction 1: F1; Fraction 2: F2). In Experiment 1, semen was frozen in egg yolk (EY) or PE extenders. After thawing, similar results were obtained in terms of total and progressive motility, viability, hypo-osmotic swelling test (HOST) and acrosome integrity after 2 h incubation. In Experiment 2, addition of rosemary to F1, F2 or both fractions to EY extenders was evaluated. Rosemary in F1 decreased progressive motility (p = 0.013) after 2 h incubation. Finally, PE can be used as a substitute for EY to reduce hygienic risks in extenders and is easier to standardize. Supplementation of EY extender with rosemary in F1 reduced progressive motility. Rosemary supplementation in F2 does not affect semen quality.

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The aim of this study was to evaluate the effects of the addition of different sugars (lactose, trehalose and glucose) on boar spermatozoa cryopreserved in an egg yolk based extender. Ejaculates were collected from a boar previously selected and semen samples were processed using the straw freezing procedure. In experiment 1, subsamples of semen were frozen in three different extenders: recommended lactose egg yolk extender (LEY); trehalose egg yolk extender (TEY) and glucose egg yolk extender (GEY). Sperm quality was assessed for motility, viability, acrosome integrity and hypoosmotic swelling test response upon collection, after freezing and thawing and then every hour for 3h. Results showed that total motility at 1 and 3h, progressive motility at 3h, positive hypoosmotic response at 2 and 3h and acrosome integrity at all times were significantly improved when trehalose was added to the extender. In experiment 2, sugar influence was also demonstrated in vitro fertilization. A total of 1691 oocytes were in vitro matured and inseminated with frozen-thawed sperm at 2000:1 sperm:oocyte ratio and coincubated for 6h. Presumptive zygotes were cultured in NCSU-23 medium to assess fertilization parameters and embryo development. Both penetration and monospermy rates were significantly higher for trehalose frozen semen. A significant increase was observed in efficiency and blastocyst formation rates from TEY to the other groups. Our results demonstrated that trehalose extender enhances spermatozoa viability and its in vitro fertilization parameters in boar ejaculates with good sperm freezability. Further studies are necessary to assess the impact of sugars on the entire population., ((c) 2010 Elsevier Inc. All rights reserved.)

Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system., ((c) 2010 Elsevier Inc. All rights reserved.)

This study was designed to evaluate the effect of adding the detergent Equex-STM to the extender used to dilute semen for cryopreservation on several indicators of sperm preservation. Two consecutive ejaculates per day were obtained from 5 Assaf sheep on two days out of every week over three alternate months. The freezing protocol involved diluting the semen in Fiser's extender, to which 0.7 percent Equex-STM was added or omitted before cryopreserving the semen in straws by exposure to nitrogen vapor. Equex-STM supplementation gave rise to significantly (p=0.05) improved sperm quality variables after different periods of freezing (0 hours, 1 week and 1 month). The variables examined were: individual motility, viability, acrosome integrity, plasma membrane integrity (HOS test) and morphological anomalies. This improvement was independent of the ram and month of testing. In a second experiment in which we incubated the semen (0 and 6 hours) at 37 degree C after thawing, Equex-STM also showed a beneficial effect on sperm quality.