Supplementation of sperm cryopreservation media with cell permeable superoxide dismutase mimetic agent (MnTE) improves goat blastocyst formation

The aim of this study was to assess whether a cell permeable superoxide dismutase agent such as MnTE, can further improve the quality of frozen/thawed semen sample using a commercially optimized sperm cryopreservation media (Bioxcell). Bioxcell was supplemented with different concentration of MnTE. Sperm membrane integrity, motility, viability and acrosomal status were assessed after freezing. Optimized concentration of MnTE was defined and used to assess fertilization and developmental potential. 0.1 μM MnTE significantly improved membrane integrity while 0.01 μM MnTE significantly improved acrosomal integrity post thawing. Addition of 0.01 μM MnTE also improved blastocyst formation rate. Supplementation of commercially optimized cryopreservation media with MnTE further improves the quality of goat frozen semen sample and may have important consequence of future embryo development. This effect may be attributed to cell permeable behavior of this antioxidant which may protect sperm genome from ROS-induced DNA damage.     

Effects of different extenders on DNA integrity of boar spermatozoa following freezing–thawing

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100 mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing–thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P < 0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100 mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100 mM trehalose, but cryopreservation could increase the degree of DNA damage (P < 0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.     

Antioxidant effects of clove bud (Syzygium aromaticum) extract used with different extenders on ram spermatozoa during cryopreservation

Clove bud (Syzygium aromaticum) extract was added at concentrations of 0, 35, 75, and 115 μg/ml to ovine semen extenders in order to investigate the antioxidant activities of clove bud extract and its effects on semen quality parameters after cryopreservation of ram spermatozoa. The basic extender was composed of Tris, egg yolk, and glycerol. Two other extenders were prepared by substitution of egg yolk with either LDL or egg yolk + SDS. The DPPH inhibition test was employed to assess the antioxidant activity of clove bud extract. Results showed that, compared to vitamin E, clove bud extract had a higher antioxidant activity. Better sperm motility and movement characteristics (P < 0.05) were observed in the semen diluted with medium containing egg yolk + SDS than in that containing egg yolk and LDL. Progressive motility and movement characteristics of the sperm were significantly improved (P < 0.05) by adding 35 and/or 75 μg/ml of clove bud extract to semen extenders. Sperm viability and plasma membrane integrity were also higher (P < 0.05) in the semen exposed to medium containing egg yolk + SDS and 75 μg of clove buds extract after cryopreservation processes. Higher levels of clove bud extract, however, had adverse effects on all the sperm quality parameters and significantly reduced (P < 0.05) the motility, movement parameters, viability, and plasma membrane integrity of ovine sperm. It was concluded that the clove bud extract had an antioxidant potential that makes it useful for addition to semen extenders and that the best results are obtained with a maximum clove bud extract of 75 μg/ml. Moreover, the combination of egg yolk and a detergent was found to improve sperm quality after the cooling and freeze–thawing processes.     

Alterations of sperm DNA integrity during cryopreservation procedure and in vitro incubation of bull semen

An association between sperm DNA integrity and fertility was recently shown for frozen–thawed Norwegian Red (NRF) bull semen diluted in skimmed milk egg yolk (SMEY). In general the fertility of NRF cattle is high, however, in comparison with NRF semen in SMEY, NRF semen diluted in Tris EY based extenders has shown reduced fertility. The aim of the present study was to do a split-sample comparison of sperm DNA integrity of NRF bull semen (n = 20) in SMEY and Triladyl^® (Tris EY based) during routine cryopreservation procedure and during in vitro incubation of frozen–thawed semen in modified synthetic oviduct fluid (mSOF). In contrast to the high fertility of NRF cattle, Holstein cattle are experiencing a marked decline in fertility. Therefore, the present study also aimed to compare sperm DNA integrity of NRF (n = 20) and Holstein (n = 20) semen diluted in Triladyl^® during in vitro incubation. The sperm DNA integrity was measured by susceptibility to in situ acid induced denaturation by the Sperm chromatin structure assay (SCSA). Compared to initial values of frozen neat semen, an increase in DNA damage was observed after dilution and cooling (5 °C) and after freezing–thawing of NRF semen in SMEY, but only after freezing–thawing for NRF semen diluted in Triladyl^®. Sperm DNA damage of NRF semen increased during in vitro incubation in mSOF; the increase in percentage of spermatozoa with DNA damage was more prominent in SMEY than in Triladyl^®, while the degree of damage was higher in Triladyl^®, throughout the incubation period. However, while the correlation between DNA damage and sperm survival was negative in SMEY throughout the incubation period, a positive correlation was observed in Triladyl^® after 9 h of incubation, indicating a higher presence of DNA damage in the live sperm population. In comparison with Holstein spermatozoa, the sperm DNA integrity of NRF semen reflected a better ability to withstand alterations induced during in vitro incubation in mSOF. In conclusion, sperm DNA integrity of NRF bull semen was altered during the cryopreservation procedure and in vitro incubation in mSOF. Dilution in Triladyl^® maintained bull sperm DNA integrity better than dilution in SMEY. Furthermore, alterations in Holstein sperm DNA integrity was more pronounced during in vitro incubation in mSOF compared to NRF bull spermatozoa.     

The protective effect of egg yolk from different avian species during the cryopreservation of Karayaka ram semen

Egg yolk is one of the most widely used cryoprotective components for sperm preservation and a wide range of factors affect its action on sperm motility, viability and fertilizing ability. The aim of this experiment was to determine the effect of different species egg yolk, namely the domestic chicken (Gallus gallus domesticus), the goose (Anatidae anser), turkey (Meleagris gallopavo), duck (Anatidae anas platyrhynchos), Japanase quail (Coturmix japonica) and chucker (Alectoris chukar) on sperm quality following cryopreservation of ram semen. Ejaculates were collected using the artificial vagina from three Karayaka rams and spermatological characteristics assessed for the pooled semen. Semen samples were evaluated as split ejaculates in the trial and samples extended with a Tris-citric acid-glucose extender containing the different avian egg yolk (15%) and glycerol (5%). The semen straws were equilibrated at 4 °C for 2 h, frozen in liquid nitrogen vapour (for 15 min at ?120 °C) and stored in liquid nitrogen (?196 °C). After thawing (37 °C for 30 s), sperm motility, viability, abnormal acrosome and membrane integrity (HOST) were evaluated. Results showed chucker egg yolk to have the best cryoprotective effect in terms of the highest sperm motility (54.0%), compared to the other five avian egg yolks (p < 0.05) evaluated. Sperm frozen in chucker egg yolk also showed a higher percentage viability (59%), than sperm stored in quail and turkey egg yolk (p < 0.05). The percentage of acrosomal abnormalities after thawing was lower in the chucker egg yolk, than the other species (p < 0.05). There was no significant difference in sperm membrane integrity between the egg yolks, except for the quail (p < 0.05). Results suggest that chucker egg yolk could be used as an alternative for chicken egg yolk, in a semen extender in cryopreservation, but it warrants further evaluation in fertility trials.     

The breeding management affects fresh and cryopreserved semen characteristics in Melopsittacus undulatus

Melopsittacus undulatus is a companion parrot worldwide diffused. Many parrots are considered endangered or vulnerable. The preservation of semen is crucial in endangered species, thus, M. undulatus could be a good model to study sperm characteristics and semen cryopreservation in these other endangered parrots. In this study the effect of the breeding management (males bred in promiscuous aviary or in couple) on sperm characteristics (motility, membrane integrity and morphometry) of fresh and cryopreserved semen was evaluated. The computer-assisted sperm analysis (CASA) revealed a significant effect of the husbandry method on semen characteristics in budgerigars: male housed in couple with the female in individual cages allowed the higher results in term of both semen quantity and sperm quality. Total and progressive motility were significantly higher in males bred in couple (68.7 ± 8.9% and 54 ± 15.9%, respectively) than in promiscuous aviary (48.3 ± 15.1% and 24.4 ± 12.4%, respectively), such as sperm velocity (average path velocity, straight line velocity, and curvilinear velocity). The type of sperm movement (amplitude of lateral head displacement, beat cross frequency, straightness, and linearity), sperm membrane integrity and morphometry parameters seemed not affected by the husbandry method. The standardization of a CASA procedure for the semen analysis in M. undulatus allow further studies on parrot semen manipulation and cryopreservation, but the method used for the breeding of the male could have a significant effect on the semen quality.     

The cryoprotective effect of low-density lipoproteins in extenders on bull spermatozoa following freezing–thawing

Egg low-density lipoprotein (LDL) was added at concentrations of 7–10% to the extenders used to freeze bull semen and its effects on the motility, mitochondria activity, acrosome integrity, membrane integrity and DNA integrity of frozen–thawed sperm were assessed. Analysis of data showed that the motility and characteristics of spermatozoa movement were higher with LDL in the extender, as compared to the extender containing 20% egg yolk. The results indicated that 8% LDL supplementation provided the highest sperm motility (55.8%) and movement characteristics (VSL, straight linear velocity: 33.8 μm/s; VCL, curvilinear velocity: 50.2 μm/s; LIN, linearity index: 56.5%; STR, mean coefficient: 76.7%; VAP, average path velocity: 35.9 μm/s; WOB, wobble coefficient: 63.9%). A concentration of 10% LDL resulted in a significant decline in the VSL, LIN, VAP and WOB values (P < 0.05). Supplementation of LDL at 8% LDL resulted in significantly higher spermatozoa mitochondrial activity, acrosome integrity, membrane integrity and DNA integrity (P < 0.05). According to all measured parameters, the extender containing 8% LDL showed beneficial cryoprotective effects on frozen–thawed bull spermatozoa. In conclusion, our results indicated that the extender containing 8% LDL extracted from egg yolk could be used successfully in the cryopreservation of bull semen with an efficacy that would be greater than present extenders containing 20% egg yolk.     

DNA integrity of fresh and frozen canine epididymal spermatozoa

The aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1 mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3 ± 3.6) and samples frozen with (4.2 ± 3.8) or without (3.6 ± 3.7) melatonin. Sperm motility was significantly (p < 0.0001) higher in fresh compared to frozen samples. The presence of melatonin in the freezing extender did not affect the sperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (p < 0.01) by cryopreservation, and melatonin did not exert any beneficial effects. In conclusion, DNA fragmentation of canine epididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques.     

The advantages of LDL (Low Density Lipoproteins) in the cryopreservation of canine semen

A medium containing LDL (Low Density Lipoproteins, the cryoprotective component of chicken egg yolk) was compared with egg yolk for the preservation canine spermatozoa during the freeze–thaw process. Twenty sperm samples taken from 10 dogs were frozen in liquid nitrogen at −196 °C in seven different media: one control medium containing 20% egg yolk, and six test media containing 4%, 5%, 6%, 7%, 8%, and 10% LDL, respectively.Following thawing, sperm motility was assessed using a Hamilton-Thorne Sperm Analyser equipped with the CEROS 12 software. The percentage of motile spermatozoa was 55.3% in the 6% LDL medium (optimal concentration) compared with 27.7% in the egg yolk based medium (p < 0.05).In comparison with the egg-yolk medium, the LDL medium also resulted in an improved preservation of spermatozoa during the freezing process (p < 0.05) in terms of acrosomal integrity (FITC-PSA test), flagellar plasma membrane integrity (HOS test), and DNA integrity (Acridine Orange test).In addition, six Beagle bitches were inseminated twice, via the intra-uterine route, at an interval of 24 h; 200 × 10⁶ spermatozoa that had been previously frozen in the 6% LDL medium were used per insemination. All of the bitches became pregnant (gestation rate of 100%).In conclusion, the 6% LDL medium provides improved protection of the spermatozoa during the freeze–thaw process and a marked improvement in the motility parameters of canine spermatozoa in comparison with the control medium containing egg yolk alone.Finally, the use of LDL as a cryoprotectant for canine semen does not interfere with fertility.     

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: Effects of extender supplemented with different antioxidants on sperm motility,velocity and fertility

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), l-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), l-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), β-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, l-methionine, SOD, l-carnitine, α-tocopherol and l-reduced glutathione (p < 0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, l-methionine, SOD, α-tocopherol and l-reduced glutathione (p < 0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.     

Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin

The process of cryopreservation impairs sperm cell function, potentially leading to a reduction in fertility. The objectives of the present study were to evaluate the effects that cryopreservation using two different extenders has on sperm motility and mitochondrial function, as well as on the integrity of plasma membranes, acrosomal membranes and chromatin, using practical and objective techniques. The focus of the present study was to identify correlations between alterations in sperm membranes and sperm motility in cryopreserved bovine spermatozoa. Seven ejaculates were collected from eight Simmental bulls (n=56). After collection, semen volume and concentration were assessed for purposes of dilution. Sperm motility was evaluated subjectively and by computer-assisted semen analysis, morphological characteristics were evaluated by differential interference microscopy, the integrity of plasma and acrosomal membranes, as well as mitochondrial function, were determined using a combination of fluorescent probes containing fluorescein isothiocyanate-Pisum sativum agglutinin, propidium iodide or 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. Chromatin integrity was evaluated using the acridine orange technique. The semen was subsequently divided into two aliquots and diluted with one of two extenders (Bioxcell or Botu-Bov), after which both were packaged in 0.5 mL straws and frozen using an automated system. Two straws of semen from each treatment were thawed, and the semen parameters were evaluated as described above. Cryopreservation of sperm reduced motility, damaging plasma and acrosomal membranes, as well as decreasing mitochondrial function. The Botu-Bov extender was more effective in preserving sperm motility and membrane integrity than was the Bioxcell extender.     

Soybean lecithin-based extender as an alternative for goat sperm cryopreservation

The aim of this study was to evaluate the effect of different concentrations of soybean lecithin (SL) in extenders for sperm goat cryopreservation. Sexually mature male Saanen goats (n = 4) were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a skim milk-based extender (control group; CG) or Tris extender supplemented with SL at different concentrations (G1 = 0.04%, SL G2 = 0.08% SL and G3 = 0.16%) for a final concentration of 240 × 10⁶ spermatozoa/mL. The semen samples were packed in straws (0.25 mL), frozen using an automated system and stored in liquid nitrogen (?196 °C). After thawing (37 °C/30 s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity, acrosome integrity and mitochondrial activity. No significant difference was observed among the experimental and control groups for all of the parameters (P > 0.05). However, even though the control group presented a significantly lower mitochondrial membrane potential compared to fresh semen (P < 0.05), the same did not occur for the extender supplemented with soybean lecithin, that is, it did not differ from fresh sperm (P > 0.05). The extender containing soybean lecithin at different concentrations preserved the sperm quality parameters in a manner similar to the conventional skim milk-based extender. Thus, it is concluded that an extender containing soybean lecithin as the lipoprotein source can be used for freezing goat semen.     

Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters,lipid peroxidation and anti-oxidant activities

Ram semen contains sufficient quantities of superoxide dismutase (SOD) and much lower concentrations of glutathione peroxidase (GSH-PX) and catalase (CAT) to prevent oxidative damage. The anti-oxidant capacity of the sperm cell is limited, due to a small cytoplasmic component, which contains these anti-oxidants to scavenge the oxidants. However, the concentration of these anti-oxidants may decrease considerably by the dilution of the semen. The aim of the present work was to study the effect of two anti-oxidants, namely, glutamine and an amino acid solution (BME) in a Tris-based extender on ram sperm parameters, lipid peroxidation and anti-oxidant capacity after the cryopreservation/thawing process. Ejaculates collected from 4 Akkaraman rams were evaluated and pooled at 37 °C. Semen samples which were diluted with the tris-based extender containing glutamine (2.5 or 5 mM), BME (13 or 26%), and no anti-oxidants (control) were cooled to 5 °C and frozen in 0.25-ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 5 mM glutamine led to higher motility rate (68.0 ± 4.4%) and hypo-osmotic swelling test (HOST) (64.1 ± 5.5%), when compared to glutamine (2.5 mM) and BME (13 and 26%) (P < 0.05). No significant differences were observed regarding sperm motility and HOST, following the supplementation of the freezing extender with glutamine 2.5 mM and BME (13 and 26%) after thawing. CAT activity remained significantly higher following the addition of glutamine 5 mM (6.4 ± 0.9 kU/g protein), compared to the other treatments (P < 0.01). The anti-oxidants at different levels were not effective in the elimination of malondialdehyde (MDA) formation and maintenance of SOD activities, when compared to the control (P < 0.05). Findings showed that glutamine (5 mM) supplementation in semen extenders, was of greater benefit to frozen–thawed ram sperm. Future efforts are needed to find the appropriate anti-oxidants and their effective concentrations to improve post-thaw sperm parameters (e.g. motility, membrane integrity, fertility) and anti-oxidant activities when frozen–thawed ram sperm is used.     

Motility and fertility of cryopreserved semen in Persian sturgeon,Acipenser persicus,stored for 30–60 min after thawing

We investigated the effect of storage times of frozen–thawed Persian sturgeon (Acipenser persicus) semen on the duration of sperm motility, percentage of motile sperm, and fertilization and hatching rates of fresh sperm and sperm stored for 0, 30, and 60 min at 4 °C post-thawing. Frozen thawed semen analyzed immediately after thawing had similar quality characteristics as fresh semen. For cryopreserved semen stored for 30 min after thawing the characteristics did not differ to fresh semen and cryopreserved semen. For cryopreserved semen stored for 60 min a significant decline in the parameters was observed. Fertilization and hatching rates were not affected by storage times of maximally 30 min of storage.     

Dynamics of sperm DNA fragmentation in domestic animals: II. The stallion

The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. This study reports on the validation of a sperm DNA fragmentation test based on the sperm chromatin dispersion test (SCD) for stallion spermatozoa and on its application to semen that was chilled (4 °C; n = 10) or frozen-thawed (n = 13). Semen samples were collected by artificial vagina and the proportion of sperm with fragmented DNA determined. Seminal plasma was then removed by centrifugation and the sperm pellet re-suspended in commercial extenders prior to being chilled or cryopreserved using standard industry protocols. Chilled semen was cooled slowly to 4 °C and stored for 1 h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 °C and analyzed for SCD after 0, 4, 6, 24 and 48 h storage. The results of this investigation revealed that there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation. However, within 1 h of incubation at 37 °C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6 h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage. While the sDFI of individual stallions at equivalent times of incubation was variable, an analysis of the rate of change of sDFI revealed no difference between stallions or the way in which the semen was preserved. In terms of sperm DNA fragmentation dynamics, the highest intensity of sperm DNA damage occurred in the first 6 h of incubation. We suggest that the SCD test can be used as a routine assessment tool for the development and refinement of preservation protocols designed to reduce stallion sperm DNA damage.     

Effect of centrifugation and sugar supplementation on the semen cryopreservation of captive collared peccaries (Tayassu tajacu)

The present study is aimed at evaluating the effect of centrifugation for seminal plasma removal and the supplementation of fructose or glucose to the Tris-based extender on the kinematic patterns of the motility parameters of frozen–thawed semen obtained from captive collared peccaries (Tayassu tajacu). Semen samples (n = 14) were collected from 10 sexually mature male collared peccaries by electroejaculation. These samples were further evaluated for parameters such as motility, vigor, sperm viability, membrane integrity, and sperm morphology. The samples were divided into four aliquots, and only two of these aliquots were centrifuged. The semen aliquots (centrifuged and raw semen samples) were diluted in Tris-based extenders supplemented with fructose or glucose. Egg yolk (20%) and glycerol (3%) were added to all the samples which were cryopreserved in liquid nitrogen and thawed at 37 °C/1 min. The frozen–thawed semen was evaluated for the same parameters described for the fresh semen. On the other hand, the kinematic motility patterns were evaluated by a computer-aided system. After thawing, it was observed that the values for the total sperm motility were around 30% for all the samples. A negative effect of centrifugation was verified for parameters such as sperm morphology, linearity, straightness, and beat cross frequency (P < 0.05). However, no differences between fructose and glucose were verified for any semen end point (P > 0.05). In conclusion, it is not recommended to centrifuge the ejaculates from collared peccaries prior to conducting the cryopreservative procedures using a Tris-based extender supplemented with fructose or glucose.     

Effect of centrifugation and sugar supplementation on the semen cryopreservation of captive collared peccaries (Tayassu tajacu)

The present study is aimed at evaluating the effect of centrifugation for seminal plasma removal and the supplementation of fructose or glucose to the Tris-based extender on the kinematic patterns of the motility parameters of frozen–thawed semen obtained from captive collared peccaries (Tayassu tajacu). Semen samples (n = 14) were collected from 10 sexually mature male collared peccaries by electroejaculation. These samples were further evaluated for parameters such as motility, vigor, sperm viability, membrane integrity, and sperm morphology. The samples were divided into four aliquots, and only two of these aliquots were centrifuged. The semen aliquots (centrifuged and raw semen samples) were diluted in Tris-based extenders supplemented with fructose or glucose. Egg yolk (20%) and glycerol (3%) were added to all the samples which were cryopreserved in liquid nitrogen and thawed at 37 °C/1 min. The frozen–thawed semen was evaluated for the same parameters described for the fresh semen. On the other hand, the kinematic motility patterns were evaluated by a computer-aided system. After thawing, it was observed that the values for the total sperm motility were around 30% for all the samples. A negative effect of centrifugation was verified for parameters such as sperm morphology, linearity, straightness, and beat cross frequency (P < 0.05). However, no differences between fructose and glucose were verified for any semen end point (P > 0.05). In conclusion, it is not recommended to centrifuge the ejaculates from collared peccaries prior to conducting the cryopreservative procedures using a Tris-based extender supplemented with fructose or glucose.     

Effect of cis-9,trans-11 and trans-10,cis-12 isomers of conjugated linoleic acid on the integrity and functionality of cryopreserved bovine spermatozoa

Plasma membranes of sperm subjected to low temperatures undergo changes in their structure and permeability. The addition of fatty acids in semen cryopreservation media may influence the sperm motility after thawing, possibly by maintaining the membrane fluidity due to their incorporation in lipid bilayers. In this work, different concentrations of the isomers cis-9,trans-11 and trans-10,cis-12 of conjugated linoleic acid (CLA) were added in the cryopreservation medium of bovine sperm. Four Jersey bulls were used, and the ejaculates were processed as a pool. The Tris-based extender (Dilutris®) was supplemented with 20% egg yolk (MB). The treatments with CLA (Luta-CLA®), which had oily presentation, were prepared from MB with addition of 1% sodium lauryl sulfate, and denominated MBL. The concentrations of CLA tested were 50, 100, and 150 μM. The motility characteristics of the post-thaw semen were analyzed by computerized analysis system (CASA), and plasma membrane integrity and acrosomal and mitochondrial function assessed by the association of the fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), JC-1 and Hoechst 33342. No significant differences were observed among treatments, excepting for a decreased mitochondrial potential of cells treated with 150 μM CLA. The addition of CLA, at the concentrations used, showed no advantages on the integrity and functionality of bovine sperm submitted to cryopreservation.     

Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling

This report describes the feasibility of using vitrification for fish sperm. Vitrification can be used to preserve samples in the field and offers an alternative to conventional cryopreservation, although it has not been systematically studied for sperm of aquatic species. The overall goal of the project was to develop streamlined protocols that could be integrated into a standardized approach for vitrification of aquatic species germplasm. The objectives of the present study in channel catfish (Ictalurus punctatus) were to: (1) evaluate the acute toxicity of 5%, 10%, 20% and 30% methanol, N,N-dimethyl acetamide, dimethyl sulfoxide, 1,2-propanediol, and methyl glycol; (2) evaluate a range of devices commonly used for cryopreservation and vitrification of mammalian sperm; (3) compare vitrification with and without cryoprotectants; (4) evaluate the post-thaw membrane integrity of sperm vitrified in different cryoprotectant solutions, and (5) evaluate the ability of vitrified sperm to fertilize eggs. Cryoprotectant concentrations of higher than 20% were found to be toxic to sperm. Methanol and methyl glycol were the least toxic at a concentration of 20% with an exposure time of less than 5 min. We evaluated a method reported for human sperm, using small volumes in loops (15 μl) or cut standard straws (20 μl) with and without cryoprotectants plunged into liquid nitrogen. Cryoprotectant-free vitrification using loops did not yield fertilization (assessed by neurulation), and the fertilization rates observed in two trials using the cut standard straws were low (∼2%). In general, fertilization values for vitrification experiments were low and the use of low concentrations of cryoprotectants yielded lower fertilization (<10%) than the use of vitrification solutions containing high cryoprotectant concentrations (as high as 25%). The highest neurulation obtained was from a mixture of three cryoprotectants (20% methanol + 10% methyl glycol + 10% propanediol) with a single-step addition. This was reflected in the flow cytometry data from which the highest membrane integrity using loops was for 20% methanol + 10% methyl glycol + 10% propanediol (∼50%). We report the first successful sperm vitrification in fish and production of offspring from vitrified sperm in channel catfish. Although the fertilization values were low, at present this technique could nevertheless be used to reconstitute lines (especially in small aquarium fishes), but it would require improvement and scaling up before being useful as a production method for large-bodied fishes such as catfish.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.