Post-thaw Motility Research Articles

Comparative Study between Slow Conventional Freezing and Cryoprotectant-Free Vitrification of Human Spermatozoa in Large Volume

Background: Cryopreservation is the collection, freezing, and long term storage of sperm, and is a highly effective method of protecting male fertility. Cryopreservation of semen has been widely used as a vital method for fertility preservation of male patients before undergoing chemotherapy, radiotherapy, and/or surgery that may lead to testicular failure or ejaculatory dysfunction. Objective: The aim of this study was to compare the aseptic technology of cryoprotectant-free vitrification of human spermatozoa in large volume, to conventional freezing protocol as regards post thawing motility, vitality and sperm DNA fragmentation. Patients and methods: This study included a total of 20 male patients seeking seminal fluid analysis, attending at the andrology laboratory of a specialized IVF center (ADAM International Hospital for Fertility and Sterility, Giza, Egypt. Patients were presented with the diagnoses of normozoospermia, oligozoospermia (either isolated or combined with asthenozoospermia or teratozoospermia). Results: Motility of (in a large volume (300 µl) in the absence of permeable cryoprotectants displayed significant statistically lower levels as compared to conventional Sperm Freezing. It was shown in different groups at different times (post thawing and 1-hour and 24-hour) of assessment that motility of vitrified spermatozoa decreases in comparison with slow conventional freezing as we go from the baseline. DNA fragmentation of vitrified spermatozoa showed higher levels as compared to conventional slow freezing but there is no significant statistical difference between vitrification and conventional slow freezing in DNA fragmentation. Conclusion: It could be concluded that vitrification technique was quite far away from comparison with slow conventional freezing protocol, and still need for further modifications and wide scale of study to achieve the good results.

Gallic and carnosic acids improve quality of frozen-thawed ram spermatozoa.

The objective was to determine effects of gallic acid (GA) and carnosic acid (CA), present in carob pods and rosemary extract respectively, on frozen-thawed ram spermatozoa. Thirty ejaculates were collected from five Merino rams, pooled, diluted in Tris-based extender and divided into five equal portions containing: 0.05 or 2mM of GA; 0.05 or 0.2mM of CA; or no additive (control). Extended semen was equilibrated at +4°C, loaded into straws, held 5cm above liquid nitrogen for 12min then plunged. Computer-aided sperm analysis was used to assess motility, whereas flow cytometry was used to assess high mitochondrial membrane potential (HMMP) and percentages of spermatozoa with plasma membrane and acrosome integrity (PMAI). Spermatozoa supplemented with 2mM GA had greater total motility than control spermatozoa (39.9±3.01 vs. 29.2±1.31%, mean±SEM, p<.05). The PMAI was greatest in 0.2mM CA (13.3±0.68%), whereas HMMP was highest in 0.05mM CA but lowest in control (22.9±4.95 and 11.4±3.64% respectively; p<.05). In conclusion, for cryopreservation of ram semen in Tris-based extender, supplementation with 2mM GA increased post-thaw motility, whereas supplementation with 0.05mM CA enhanced mitochondrial function.

Effects of cryopreservation on cAMP-dependent protein kinase and AMP-activated protein kinase in Atlantic salmon (Salmo salar) spermatozoa: Relation with post-thaw motility

Sperm motility in fish with external fertilization is critical for reproductive efficiency in aquaculture, especially in salmonids. Gamete preservation techniques, such as cryopreservation, however, reduce sperm motility and fertilizing capacity. Very few studies have addressed cryodamage from energetic and cell signalling approaches. In this study, cAMP-dependent protein kinase (PKA) and AMP-activated kinase (AMPK) activities were quantified in fresh and cryopreserved spermatozoa of Atlantic salmon (Salmo salar); and the relation with motility was analysed. Results indicate there was a decrease in membrane integrity and motility in post-thawed spermatozoa compared to fresh samples, however, there was about 30% of cells with intact plasma membrane but incapable of motility. The PKA and AMPK activities were less after cryopreservation, indicating that loss of motility may be related to alteration of these key enzymes. Furthermore, PKA and AMPK activities were positively correlated with each other and with motility; and inhibition decreased motility, indicating there is a functional relationship between PKA and AMPK. The PKA inhibition also decreased AMPK activity, but results from protein-protein docking analyses indicated AMPK activation directly by PKA is unlikely, thus an indirect mechanism may exist. There have been no previous reports of these kinase actions in fish spermatozoa, making these findings worthy of assessment when there are future studies being planned, and may serve as base knowledge for optimization of cryopreservation procedures and development of biotechnologies to improve reproduction efficiency in the aquaculture industry.

Effect of extender, cryoprotectants and GameteGuard® on the post-thaw motility in boar sperm

Effect of extender, cryoprotectants and GameteGuard® on the post-thaw motility in boar sperm

Differential motility parameters and identification of proteomic profiles of human sperm cryopreserved with cryostraw and cryovial

BackgroundAlthough sperm cryopreservation has been widely used in human reproductive medicine as an integral infertility management in infertility clinics and for banking sperm in sperm banks, the freezing/thawing protocols are not optimal. The freezing and thawing processes result in changes at both structural and molecular levels, some even detrimental, in human sperm when compared with fresh sperm. The change of sperm proteins after cryopreservation may play negative roles for fertilization and early embryo development. Conventionally, cryostraws (CS) and cryovials (CV) are the most widely used cryopreservation carriers (CPCs) for human sperm cryopreservation accompanied with the use of egg yolk free commercial media. However, the influence of cryopreservation on the proteomic profile of human sperm preserved with the two CPCs is unknown. Therefore the purpose of the present study was to compare the frozen-thawed motility, investigate the proteomic profile of human sperm cryopreserved with the two types of CPCs, and identify the susceptible proteins that play key roles for sperm function and fertility.MethodsThe present study compared the cryosurvival of human sperm frozen with the two different CPCs and identified the sperm proteomic changes by using the isobaric tags for relative and absolute quantification labeling technique coupled with 2D LC–MS/MS analysis after freezing and thawing.ResultsOur results indicated that sperm cryopreserved with CV showed higher values for percentage of motile sperm and forward activity rate than those with CS. Compared to fresh sperm, 434 and 432 proteins were differentially identified in human sperm cryopreserved with CS and CV, respectively.ConclusionThe proteomic profiles of human sperm are greatly affected by cryopreservation with either type of CPC. GO analysis revealed that most of the differentially identified sperm proteins enriched in the extracellular membrane-bounded organelles, cytoplasm and cytosol. In addition, 106 susceptible proteins having known identities related to sperm functions were identified. In general, cryovial seems to be the preferred CPC for human sperm cryopreservation based on the post-thaw motility parameters and the effect on sperm proteomic profiles. These results are beneficial for the insight into the understanding of the cryoinjury mechanism of sperm and the development of human sperm cryopreservation strategies.

Effects of extender, cryoprotectants and thawing protocol on motility of frozen-thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses

We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose–EDTA–egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk–egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially thawed to 5 °C, diluted and refrozen in SMEY containing 2, 4, 6 or 8% GLY or GMF. In Experiment 1, sperm motility was reduced after each cryopreservation cycle (P < 0.05). Extender type did not affect motility after refreezing (P > 0.05), but sperm initially thawed to 5 °C exhibited higher motility than sperm thawed to 37 °C (P < 0.05). In addition, sperm refrozen in SMEY containing MF or GMF exhibited higher motility than sperm refrozen in GLY alone (P < 0.05). In experiment 2, there was an interaction between CPA and CPA concentration (P < 0.05). Sperm refrozen with GMF had higher motility than refrozen sperm with GLY (P < 0.05), and while GLY concentration did not affect post-thaw motility (P > 0.05). Sperm refrozen with 6 or 8% GMF exhibited the highest motility (P < 0.05). In conclusion, sperm motility is best maintained when thawing and refreezing stallion sperm in low sperm concentration ICSI doses by initially thawing the sperm to 5 °C and diluting the sperm in a freezing extender with 8% GMF.

Copy number variation of ZNF280AY across 21 cattle breeds and its association with the reproductive traits of Holstein and Simmental bulls

The mammalian Y chromosome gene families in the ampliconic region are expressed predominantly or exclusively in the testis, and their copy number variations (CNV) are significantly associated with male reproductive traits, suggesting they have important roles in spermatogenesis and testicular development. ZNF280AY (zinc finger protein 280A, Y-linked) is a member of the zinc finger protein family and has been identified as a bovid-specific Y-chromosome gene. The current study applied a reliable quantitative real-time PCR method to estimate the CNV of ZNF280AY in 715 bulls across 21 cattle breeds and to further investigate the association of the CNV of ZNF280AY with bull reproductive traits and ZNF280AY mRNA expression levels in adult testis. The results revealed that the median copy number of ZNF280AY was 47, and the copy number varied from 11 to 154, showing significant CNV between and within the investigated cattle breeds. In addition, all 715 bulls were classified into Y1, Y2, and Y3 lineage groups based on a rapid genotyping method described previously. Pairwise comparisons indicated that bulls belonging to the Y1 lineage had a significantly lower median copy number (40) than bulls belonging to the Y2 (52) and Y3 lineages (57). Association analysis revealed that the CNV of ZNF280AY was correlated negatively with the percentage of normal sperm and sperm concentration in Holstein bulls, whereas no significant correlation was observed with ejaculation volume, total sperm count, sperm motility, postthaw motility (PTM), and scrotal circumference in Holstein and Simmental bulls. Furthermore, no correlation was observed between ZNF280AY copy number and ZNF280AY mRNA expression levels in the testis. The current study suggests that the CNV of the ZNF280AY gene family is associated with male reproductive traits and may serve as a valuable marker for early bull fertility selection in Holstein breeding programs.

Collection and cryopreservation of epididymal spermatozoa in dogs

When a male dog is not able to ejaculate, epididymal spermatozoa can be used to artificially inseminatethe bitch. In the present study, two methods to collect epididymal sperm after castration werecompared, i.e. the float-up method and the aspiration method. With the float-up method, more spermatozoawere collected than with the aspiration method (1810±718.1 million versus 694±244.5 millionrespectively, p-value = 0.018). A trend towards a higher post-thaw motility was observed when usingthe float-up method compared to the aspiration method (52±9.4% versus 44±9.4%, p-value = 0.060).Semen samples collected with the float-up method showed more blood contamination than with theaspiration method; however, this seemed to have no effect on the quality and quantity of the semen. Itcan therefore be concluded that the float-up method is the method of choice when epididymal spermhas to be collected in dogs.

Interactions of ostrich egg yolk lipoproteins with seminal plasma of fractionated ejaculates and their effects on post-thaw boar semen quality.

Electrophoretic methods were used to identify protein complexes formed between ostrich egg yolk lipoprotein fractions (LPFo) with seminal plasma (SP) of fractionated ejaculates, and to investigate the effect of these complexes on boar semen quality after cryopreservation. Chromatographic SP fractions (F1, F2 and F3), with or without LPFo solution, were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Comparative electrophoretic analyses of the SP revealed marked differences in the SDS-PAGE protein profiles among boars. Electrophoretic analyses showed that the interactions of LPFo with SP resulted in the appearance of high-intensity protein bands. Spermatozoa were exposed to SP chromatographic fractions originating from F1, F2 and F3, and the whole SP (wSP) before being frozen. Spermatozoa exposed to F1 and F2 exhibited significantly higher post-thaw motility compared to those treated with either F3 or wSP. In most of the boars the proportions of membrane- -intact frozen-thawed spermatozoa differed among the treatments, being significantly lower in the wSP-treated samples. The incidence of frozen-thawed spermatozoa with DNA fragmentation was less prevalent in samples exposed to F3 or the wSP. The results of this study confirmed that the interactions of LPFo with fractionated SP during the cooling period contributed to alterations in the sperm membranes, rendering them less susceptible to temperature-related injury.

Cryopreservation and hormonal induction of spermic urine in a novel species: The smooth-sided toad (Rhaebo guttatus)

Global amphibian declines have fueled an increased interest in amphibian assisted reproductive technologies. Within the genus Rhaebo, half of the species are experiencing decreasing population trends; however, insufficient information is available on many of these species’ reproductive biology. Using the smooth-sided toad, Rhaebo guttatus, we present effective methods for collecting and cryopreserving an example of Rhaebo sperm. Specifically, our findings show that administering 10 IU/g body weight of hCG (human chorionic gonadotropin) yields the most motile and concentrated sperm and that cryopreserving spermic urine in a solution of 5% DMFA (N,N-Dimethylformamide) and 10% trehalose returns sperm with a 33 ± 3% average post-thaw motility. These findings may represent an important step forward in developing techniques that can be safely applied to other, more vulnerable species within the Rhaebo genus.

Staalname en cryopreservatie van epididymale spermatozoa bij de hond

Wanneer een reu niet kan ejaculeren, kan epididymaal sperma gebruikt worden om een teef kunstmatig te insemineren. In deze studie werden twee methoden vergeleken om epididymaal sperma te verzamelen na castratie, namelijk de “float-up”-methode en de aspiratiemethode. Met de float-up-methode konden meer spermatozoa verzameld worden dan met de aspiratiemethode (1810±718,1 miljoen versus 694±244,5 miljoen respectievelijk, p-waarde = 0,018). Wanneer sperma verzameld werd met de float-up-methode, kon na het invriezen een trend tot een hogere motiliteit waargenomen worden dan met de aspiratiemethode (52±9,4% versus 44±9,4% respectievelijk, p-waarde = 0,060). Deze stalen vertoonden meer bloedcontaminatie dan de stalen verzameld met de aspiratiemethode. Dit bleek echter geen nadelig effect te hebben op de spermakwaliteit en -kwantiteit. Daarom kon geconcludeerd worden dat bij voorkeur de float-up-methode dient gebruikt te worden, wanneer epididymaal sperma verzameld moet worden bij honden.

Phospholipase C zeta 1 mRNA as a marker of oocyte-activation and fertilization potential of water buffalo (Bubalus bubalis) semen

The present study aimed to detect and quantify the expression of buffalo PLCZ1 mRNA (buPLCZ1 mRNA) in buffalo semen and to determine its oocyte-activation and fertilizing ability through IVF. Buffalo semen samples were collected and subjected to standard subjective physical and microscopic evaluation of semen characteristics. Small amount of fresh semen sample was taken for molecular experiment. The rest of the samples were processed into frozen semen. Both fresh and frozen semen were subjected to Reverse Transcriptase quantitative PCR (RT-qPCR) technique to determine and quantify buPLCZ1 mRNA expression. Subsequently, 40 bulls with known buPLCZ1 mRNA content were evaluated for its oocyte-activation activity through IVF using frozen semen. Correlation analysis was done to determine the relationship between the expression of buPLCZ1 mRNA and percentage of IVF, and other semen variables. PLCZ1 mRNA expression in buffalo semen was observed variable among donor bulls, but was not significantly different (P > 0.05) between fresh and frozen semen. The result suggests that the cryopreservation procedure does not affect the expression of buPLCZ1 mRNA in semen. Statistical analysis showed that the expression of buPLCZ1 mRNA in fresh semen was strongly correlated with that of frozen semen (r = 0.992; P < 0.001). Similarly, expression of buPLCZ1 mRNA in frozen semen was positively correlated with percentage of IVF (r = 0.776; P < 0.001), but not with Initial (r = −0718; P > 0.05) nor with Post-Thaw Motility (r = −0.0313; P > 0.05). A strong relationship between buPLCZ1 mRNA concentration and IVF rate indicates that buPLCZ1 mRNA concentration can be used to assess oocyte-activation and fertilizing potential of the buffalo semen. The present study essentially demonstrated buPLCZ1 mRNA as biological marker for male fertility and that molecular technique serves as an objective approach of semen quality evaluation to enhance bull selection for genetic improvement in water buffaloes.

European eel sperm storage: Optimization of short-term protocols and cryopreservation of large volumes

Maturation in captivity of European eel (Anguilla anguilla) requires long and costly hormonal treatments that often lead to asynchronic maturation between sexes. Therefore, optimization of sperm short-term storage methods and cryopreservation protocols can be a key factor for successful artificial fertilization. Two experiments were carried out to optimize the existing protocols.For the short-term storage experiment, sperm was diluted in P1 extender and then stored at different dilution ratios (1,9 and 1,49). The best outcome was then tested at different temperatures (4 and 20 °C) and in constant agitation or still. In the cryopreservation experiments, large sperm volumes (cryotubes of 2 and 5 ml), different cooling rates (freezing tubes 1 or 3 cm above liquid nitrogen during 15 and 20 min), and different extender compositions (methanol 10% was used as cryoprotectant, and complemented with FBS 20%, BSA 5% or egg yolk 5%) were tested. Sperm kinetic parameters were analyzed with a CASA-Mot system both in fresh and short- or long-term stored samples.In the short-term storage trial, sperm quality did not show significant differences in the first 24 h after sperm collection between the different storage conditions tested. For longer time, 1:49 dilution ratio showed significantly better results than 1:9, and low temperature (4 °C) was better for sperm preservation after 3 days.Cryopreserved sperm samples showed good motility results when they were frozen in cryotubes of 2 and 5 ml, with no significant differences compared to samples cryopreserved in lower volumes (straws of 0.5 mL). Furthermore, the combination of methanol (10%) and egg yolk (5%) as freezing medium, induced significant higher post-thawing motility values (over 50%) than the control (methanol 10%), whereas the addition of FBS (20%) and BSA (5%) led to a significant reduction of the sperm motility. The establishment of these storage and cryopreservation protocols will be important for the improvement of European eel artificial reproduction programs.

Effect of Different Extenders and Dilution Ratios on the Spermatological and Fertilization Parameters of Cyprinus carpio During Cryopreservation.

Production of quality seeds is a critical need, and cryopreservation serves as an important biotechnological tool in achieving that goal. In the present study, the effects of different extenders and dilution ratios on the spermatological and fertilization parameters of cryopreserved milt were evaluated in common carp brooders. Milt was collected from Cyprinus carpio brooders and diluted and cryopreserved at three dilution ratios such as 1:40, 1:80, and 1:120, using Freshwater Fish Saline (FWFS) and Modified Fish Ringer as extenders and dimethyl sulfoxide as cryoprotectant (90:10). Motility duration, fertilization, and hatching rates were studied after different treatments. Rapid freezing of straws was done for 20 minutes at a freezing rate of -78°C/min. It can be concluded that C. carpio milt can be cryopreserved with FWFS at a dilution ratio of 1:40 to obtain the highest post-thaw motility duration (60.33 ± 1.52 seconds), fertilization rate (87.6% ± 1.52%), and hatching rate (57.1% ± 4.16%). The difference in the values between extenders, dilution ratios, and cryopreserved and fresh milt were statistically significant (p < 0.05) when analyzed using ANOVA.

Post-thaw Addition of Caffeine and/or Pentoxifylline Affect Differently Motility of Horse and Donkey-Cryopreserved Spermatozoa

To increase sperm motility, several molecules have been tested in mammals. Methylxanthines have shown effects on sperm motility, capacitation, and on in vitro fertilization processes. The aim of the study was to evaluate if the post-thaw addition of caffeine and/or pentoxifylline changes motility parameters of cryopreserved stallion and donkey spermatozoa. Straws derived from 14 horses and 7 donkeys were thawed and diluted in a milk-based extender to obtain the following final concentrations: CTR (control, no additives), CAF 5 (5 mM caffeine), CAF 10 (10 mM caffeine), PTX 5 (5 mM pentoxifylline), PTX 10 (10 mM pentoxifylline), CAF-PTX (5 mM caffeine and 5 mM pentoxifylline). Samples were evaluated immediately post-thaw and after 60 and 120 minutes of incubation at 37°C. In horses, overall total motility was significantly lower in CTR than in to CAF5, CAF-PTX, PTX5, PTX10, whereas progressive motility increased only in CAF5 and PTX5 (P < .05). No differences between control and treatments were seen for donkey semen. In CTR, during the first hour of incubation horses' sperm cells showed a larger decrease than donkeys’ ones in all parameters (P < .05), except for lateral sperm head displacement. Thus, post-thaw motility and velocity decreased more sharply in horses than in donkeys. Caffeine and pentoxifylline-added post-thaw were able to increase the proportion of motile spermatozoa only for stallions and not for donkeys. Whether the improvement in post-thaw motility of equine spermatozoa may have an effect on in vivo or in vitro pregnancy rates remains to be determined.

Effect of Dietary Supplementation of Trace Minerals on Semen Production Performance of Sahiwal Bulls During Winter Season

Present study was planned to evaluate the effect of supplementaltrace mineralson semen production performance of Sahiwal bulls. Twelve Sahiwal bulls were grouped as T 1 , T 2 and T 3 based on their semen volume and concentration and were fed with 0, 1.5 and 2.0 times higher trace mineralsover and above the basal diet in T 1 , T 2 and T 3, respectively for a study periodof 120 days. Semen collection was done twice a week in all the groupsand samples were evaluated fortnightly forquantitative and qualitative attributes. A seven days digestion trial was conducted at 90 d of experimental feeding to assess the digestibility of nutrients. Alkaline phosphatase (ALP) activity, lipid peroxidation (LPO) activity,blood antioxidant status and mineral (Zn, Cu, Mn, Co, Fe, Ca and P) estimation was done inblood and seminal plasma. It was found that additionaltrace minerals supplementation had no influence on body weight, dry matter intake and digestibility [dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fibre (NDF) and acid detergent fibre(ADF)] among the groups. All seminal parameters except mass activity and post thaw motility were higher ( P <0.05) in T 3 group in comparison to T 1 and T 2 groups in fresh as well as frozen semen. Blood antioxidant status was improved in T 3 group. A reduction ( P <0.05) in LPOactivityin fresh and frozen semen in T 3 group was observed. It may be concluded that dietary supplementation of additional Zn, Cu, Mn and Co (40 ppm Zn, 10 ppm Cu, 15ppm Mn and 0.11ppm Co)over and above the basal diet per day per Sahiwal bull can improve the qualitative and quantitative attributes of semen.

Recent advances in cryopreservation of semen and artificial insemination in sheep: A review

Cryopreservation of semen facilitates widespread dissemination of superior germplasm and thus allowing the full exploitation of artificial insemination (AI) benefits. Recent focus is on preserving fertilizing capacity of frozen spermatozoa by improving the composition of extenders and changing cooling/freezing protocols, use of cryoprotectants, antioxidants, including enzymes, vitamins, amino acids and proteins, plant extracts and other compounds. In addition, research is also intensified on role of seminal plasma and its proteins as well as cholesterol-loaded cyclodextrin in semen freezing. Recently it has also been shown that helium-neon laser irradiation of cryopreserved ram sperm increased the post-thaw sperm motility, viability, cytochrome c oxidase activity and ATP contents with no significant changes in DNA and acrosome integrity. Post-thaw sperm motility of cryopreserved semen ranged from 33.3 to 72.2%. Fertility depends on the insemination technique and ranged from 45 to 80% for laparoscopic and 4 to 42% for transcervical insemination. Further, the complex anatomy of ewe's cervix is also a hurdle affecting intrauterine deposition of semen and consequently the fertility of frozen-thawed semen. Researchers are attempting cervical dilation with the use of hormones and chemicals to facilitate catheter penetration in the cervical canal. This review gives a detailed semen processing techniques and their effects on semen quality and AI techniques used in sheep and their success rates.

Bovine Sperm Motility as Affected by Alpha Tocopherol and Ascorbic Acid during Storage

Our purpose was to determine the impact of ascorbic acid and α-tocopherol on bovine sperm motility following short-term storage or cryopreservation. Semen was collected from mature Angus bulls (n = 4). The experimental design was a randomized complete block, with bull serving as block, and treatments were structured as a 4 × 4 factorial with four concentrations of ascorbic acid (0, 5, 10, 20 mM) and four concentrations of α-tocopherol (0, 0.05, 0.5, 5 mM). Sperm motility characteristics were evaluated using computer assisted sperm analyses at 0, 4, and 8 h of incubation (39˚C) and post-cryopreservation. Initial sperm motility was (79.6% ± 1.6) and decreased to (6.1% ± 1.6%) after cryopreservation. Cryopreserved spermatozoa had lower (P 0.1) by α-tocopherol. Addition of 20 mM ascorbic acid to storage media decreased (P < 0.05) sperm velocity traits, but the addition of 5 or 10 mM ascorbic acid did not alter sperm velocity. Sperm cell oxidation following cryopreservation/post-thaw was affected by an interaction (P < 0.05) between concentrations of ascorbic acid and α-tocopherol. Stepwise regression models predicted (P < 0.05) post-thaw motility and velocity characteristics of cryopreserved spermatozoa. Our results suggest that adding ascorbic acid and α-tocopherol was not beneficial for short-term storage of spermatozoa; however, our results were inconclusive with regards to inclusion of ascorbic acid and α-tocopherol in egg yolk-based cryopreservation media.

Influence of addition of cobalamin to the extender on the post-thaw motility, viability, and DNA integrity of bovine ejaculate

The present study was undertaken to investigate the effect of various concentrations of cobalamin (vitamin B12) in Bioexcell® extender on the spermatozoa quality of Simmental bulls. The semen was collected from 12 bulls by means of an artificial vagina. Vitamin B&lt;sub&gt;12&lt;/sub&gt; at concentrations of 1.50, 2.50, 3.50, and 5.00 mg/mL was added to bovine Bioexcell® extender. The semen aliquots were cooled and preserved at 4°C. Their quality was evaluated during pre-freezing, and then the cooled semen samples were packaged into 0.25 ml straws. The straws were frozen in the vapor of liquid nitrogen, and stored at -196ºC in a container. The straws were thawed one day later, and the characteristics of spermatozoa were examined. The results showed that the effect of vitamin B&lt;sub&gt;12&lt;/sub&gt; on the viability, DNA fragmentation and motility of spermatozoa was significant under both pre- and post-freezing conditions (p &amp;lt;0.05), and revealed that supplementation of the extender with B&lt;sub&gt;12&lt;/sub&gt; improved the post-thaw spermatozoa quality in bulls.

Reduced cytochrome oxidase activity and increased protein tyrosine phosphorylation of mitochondria-rich fractions of buffalo (Bubalus bubalis) spermatozoa after a cycle of freezing and thawing.

The motility and fertility of mammalian spermatozoa are compromised when they are cryopreserved. Sperm mitochondrial proteins play a vital role in conferring motility. However, the effects of cryopreservation on mitochondria-specific proteins remain primarily unexplored in domestic animals, including buffaloes, so the present study aimed to evaluate this issue. Mitochondria were isolated from both non-cryopreserved and cryopreserved buffalo spermatozoa by sonication followed by sucrose density gradient ultracentrifugation. The purity of the mitochondrial preparation was assessed by cytochrome oxidase assay and electron microscopy. Mitochondria separated from cryopreserved buffalo spermatozoa were associated with significantly lower (P ≤ 0.05) cytochrome oxidase activity as compared with non-cryopreserved spermatozoa. The intensities of two low-molecular-mass mitochondrial proteins (30.1 kDa and 26.1 kDa) were significantly reduced as compared with the non-cryopreserved group. In addition, in cryopreserved buffalo sperm mitochondria, the intensities of three tyrosine phosphorylated proteins (126.6, 106.7 and 26 kDa) increased significantly compared with the non-cryopreserved group. Of these, tyrosine phosphorylation of the 26-kDa mitochondrial protein of cryopreserved sperm was very intense and unique because it could not be detected in the mitochondria of non-cryopreserved sperm. Thus, the study confirmed that both cytochrome oxidase activity and the proteins of buffalo sperm mitochondria undergo significant cryogenic changes in terms of quantity and quality after a cycle of freezing and thawing and this may be one of the important causes of reduced post-thaw motility and fertility of cryopreserved buffalo spermatozoa.