Flow cytometric assessment of fresh and frozen-thawed Canada goose (Branta canadensis) semen

The present study was conducted to investigate spermatozoal membrane integrity, acrosome integrity, mitochondrial activity, and chromatin structure in fresh and frozen-thawed Canada goose (Branta canadensis) semen with the use of the flow cytometry. The experiment was carried out on ten, 2-year-old, Canada goose ganders. The semen was collected twice a week, by a dorso-abdominal massage method, then pooled and subjected to cryopreservation in straws, in a programmable freezing unit with the use of dimethyloformamide (DMF) as a cryoprotectant. Frozen samples were thawed in a water bath at 60 °C. The freezing procedure was performed ten times. For the cytometric analysis the fresh and the frozen-thawed semen was extended with EK extender to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with SYBR-14 and propidium iodide (PI), acrosomal damage was evaluated with the use of PNA-Alexa Fluor^®488 conjugate, mitochondrial activity was estimated with Rhodamine 123 (R123), and spermatozoal DNA integrity was measured by the sperm chromatin structure assay (SCSA). The cryopreservation of Canada goose semen significantly decreased the percentage of live cells, from 76.3 to 50.4% (P < 0.01). Moreover, we observed the significant decrease in the percentage of live spermatozoa with intact acrosomes (P < 0.01), but we did not detect significant changes in the percentage of live spermatozoa with ruptured acrosomes. However, after thawing 50% of Canada goose live spermatozoa retained intact acrosomes. Furthermore, the percentage of live spermatozoa with active mitochondria was significantly lower in the frozen-thawed semen than in the fresh semen (P < 0.05). Nevertheless, after thawing the mitochondria remained active in almost 50% of live cells. In the present study, we observed no changes in the percentage of sperm with fragmented DNA after freezing-thawing of Canada goose semen. In conclusion, the present study indicates that even the fresh Branta canadensis semen might have poor quality, the cryopreservation of its semen did not provoke spermatozoal DNA defragmentation and half of the spermatozoa retained intact acrosomes and active mitochondria after freezing-thawing.     

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.     

The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability

The Sperm Class Analyzer was used to investigate the effect of freeze-thawing procedure on Florida buck sperm head morphometry, and to relate possible changes in sperm head dimensions to cryopreservation success. Semen samples (n=76) were frozen with tris and milk-based extenders and thawed. Sperm quality samples (motility, morphology, acrosome), and sperm head morphometric values (length, width, area, perimeter, ellipticity) were compared between fresh and frozen-thawed samples. Sperm freezability was judged according to the sperm quality parameters assessed. Fertility data was obtained after artificial insemination with cryopreserved semen. Cryopreservation success was different between freezing methods. Sperm head dimensions were significantly (p<0.05) smaller in cryopreserved tris and milk spermatozoa respectively than in those of the fresh samples. The sperm head morphometric parameters that had changed after cryopreservation were lower in suitable semen samples after thawing and with successful pregnancies after artificial insemination. These data suggest that changes in sperm head morphometry might reflect spermatozoa injury occurred during cryopreservation.     

Effects of mint,thyme, and curcumin extract nanoformulations on the sperm quality,apoptosis, chromatin decondensation,enzyme activity,and oxidative status of cryopreserved goat semen

Goat semen cryopreservation is a challenging process as it results in reduced motility, vitality, and fertility of spermatozoa after freezing. In this study, we evaluated the effects of different herbal extract nanoformulations (NFs) [mint (MENFs), thyme (TENFs), and curcumin (CENFs)], supplemented at either 50 or 100 μg into Tris-extender on the cryopreserved goat semen quality. The hydrothermal squeezing method was used for the preparation of the NFs extracts. The morphological evaluation of the NFs extracts was conducted by transmission electron microscopy. All NFs supplements improved (p < 0.05) the progressive motility, vitality, and plasma membrane integrity of sperm compared with the control extender after equilibration (5 °C for 2 h) and thawing (37 °C for 30 s), but had no effect on sperm abnormality and acrosome integrity. All NFs supplements decreased (p < 0.05) the apoptosis, malondialdehyde level, and chromatin decondensation of sperm cells, while increased (p < 0.05) the total antioxidant capacity and catalase activity in the frozen/thawed extender. Particularly, CENFs at a level of 100 μg showed improvement of sperm parameters and antioxidant status during cryopreservation of goat semen more than TENFs and MENFs. The CENFs improved the quality of goat spermatozoa in post-thawed semen in terms of preventing cryodamage and promoting the cryotolerance of spermatozoa when compared with TENFs and MENFs. Therefore, supplementation of Tris-extender with CENFs could enhance goat semen processing during cryopreservation.     

Cryopreservation of semen from endangered pheasants: the first step towards a cryobank for endangered avian species

In order to improve the genetic management of bird species within the European Endangered Programs (EEP), a research project on artificial insemination and cryopreservation of Galliformes semen has been developed. The aim of the program is to create a sperm cryobank for threatened bird species. During this study, semen was collected from 17 pheasant species and specific characteristics of ejaculates were analyzed (volume, sperm concentration, motility, pH). Artificial insemination with fresh semen was performed in nine species and with frozen semen in eight species. Inseminations with frozen and thawed semen were made in 17 species. Viability of fresh and frozen semen was assessed in vitro using double stains, eosin and nigrosin. The effect of pH (7-8.5) on viability of fresh and frozen/thawed spermatozoa was also studied. Chicks hatched in eight and three species after insemination with fresh and frozen/thawed semen, respectively. Species varied widely in semen viability: 1-30% of spermatozoa survived freezing and thawing. There was a negative correlation between the viability of frozen spermatozoa and semen pH. In our experimental conditions, the pH of diluents had no effect on semen viability. However, semen with the highest pH had the lowest quality after freezing and thawing. These experiments demonstrated the feasibility of using a very simple and inexpensive method to achieve artificial insemination and cryopreservation of semen in endangered pheasant species.     

Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm

Evaluating cryoinjury of canine spermatozoa is crucial to improving the probability of fertilization. Recently, studies on sperm ROS production, phospholipid scrambling, and DNA damage induced by cryopreservation have been reported. However, the consequences of cryopreservation on these crucial factors are lacking with respect to canine semen. Therefore, the current study was designed to investigate the effects of the freezing-thawing procedure on these factors in canine semen. Ejaculates from five dogs were cryopreserved and thawed. Spermatozoa before and after a freezing-thawing process were assessed for phosphatidylserine (PS) translocation (Annexin V [AN]/propidium iodide [PI] assay), intracellular H₂O₂ level (dichlorofluorescein [DCF]/PI assay), DNA integrity (sperm chromatin structure assay), and conventional sperm parameters. The freezing-thawing process decreased motility, viability, normal morphology, and membrane integrity in canine sperm (P < 0.05). The frozen-thawed semen also showed a decrease in AN−/PI− sperm (%) and an increase in the PS translocation index, the intracellular H₂O₂ level in the viable sperm fraction, and the DNA fragmentation compared with that of fresh semen (P < 0.05). In conclusion, the freezing-thawing procedure significantly affects PS translocation, the intracellular H₂O₂ level, and DNA integrity in canine semen, which may explain the lower fertilization rate and in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) outcome when frozen-thawed spermatozoa are used. It is therefore recommended that these parameters be used as an additional parameter for the assessment of sperm quality after freeze-thawing in canine semen.     

Assessment of the efficacy of Sephadex G-15 filtration of bovine spermatozoa for cryopreservation

Semen from five dairy AI bulls was split-filtered through a Sephadex G-15 filter and frozen in a Tris-citric acid buffer egg yolk-based extender. The effect of filtration was studied morphologically for individual sperm abnormalities. Computer-assisted sperm analysis (CASA) was used for motility and sperm motion assessment. Flow cytometry was used to disclose sperm viability (SYBR-14/PI), mitochondrial membrane potential (Mitotracker Deep Red/SYBR 14), acrosome integrity (SYBR 14/PE-PNA/PI), plasma membrane stability (Merocyanine 540/YO-PRO 1/Hoechst 333342), and chromatin stability (acridine orange staining). Filtration significantly reduced the concentration of recovered spermatozoa (P < 0.01), but improved semen quality, reducing the number of spermatozoa with various forms of morphological defects. Filtration also affected percentages of sperm motility after equilibration and after freezing/thawing. Sperm motion characteristics were, however, not significantly affected by filtration at any stage of the cryopreservation protocol, including post-extension, equilibration, or freezing/thawing. Filtration enhanced sperm viability after thawing (P < 0.05), but had no significant effect (P > 0.05) on recovery of spermatozoa with high mitochondrial potential, intact acrosomes, or preserved sperm chromatin structure. Sperm plasma membrane stability was also not affected by the filtration method used (P > 0.05). It can be concluded that filtration effectively separates weaken or abnormal spermatozoa in pre-freezing semen samples and therefore the procedure could be recommended to improve post-thaw sperm viability of selected, fertile sires.     

Ginger and echinacea extracts improve the quality and fertility potential of frozen-thawed ram epididymal spermatozoa

The current study examined the impact of the supplementation of ginger and echinacea extract, as natural antioxidant agents, in freezing extender on the quality and fertility potential of ram epididymal spermatozoa after cryopreservation. Epididymal spermatozoa isolated from Forty testicles, obtained from 20 rams, with motility >80% and total morphological abnormalities <10% were pooled, divided into 7 aliquots and used for cryopreservation. The semen samples were re-suspended with basic Tris egg yolk diluent containing ginger and echinacea extracts (5, 10 and 20 mg/l). The control diluent comprised of only extender and lacked any antioxidant agent. For the determination of sperm quality, frozen straws were thawed after 7–10 days, and then the sperm characteristics were assessed. The supplementation of ginger at a concentration of 10 mg/l, as well as the addition of 10 and 20 mg/l echinacea extract significantly improved total motility and velocity parameters. The status of acrosome integrity and lipid peroxidation significantly improved in spermatozoa when supplemented with 10 mg/l ginger and 20 mg/l echinacea extract. Also, 5 mg/l ginger extract and 20 mg/l echinacea extract significantly improved mitochondrial activity. The highest ratio of the dispersion of sperm chromatin was observed in spermatozoa treated with 10 mg/l ginger extract. The cleavage rate was markedly higher in matured oocytes that were fertilized with frozen spermatozoa treated with 20 mg/l ginger extract and 10 mg/l echinacea. The application of ginger and echinacea extract resulted in improvement in the quality and fertility of frozen-thawed spermatozoa. However, future studies are wanted to elucidate how the active components in these extracts prevent cryo-damages in spermatozoa.     

Effect of oviductal proteins on sperm functions and lipid peroxidation levels during cryopreservation in buffaloes

A study was undertaken to find out the effect of addition of oviductal proteins on sperm functions and lipid peroxidation (LPO) levels in buffaloes. Oviductal flushings were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle), centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal and luteal oviductal fluid were precipitated overnight using ammonium sulphate, centrifuged (10,000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored frozen at -20 degrees C. Six pooled good quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was split into three parts and extended in Tris-Egg yolk-Citrate extender (20% egg yolk: 7% glycerol), so that final dilution yielded approximately 60 million sperm cells/ml and cryopreserved in 0.5 ml French straws (30 million sperm cells per straw) in LN2 (-196 degrees C). Before freezing, the nonluteal and luteal oviductal proteins (NLOP &LOP) were incorporated at the concentration of 1mg/ml of extended semen. The equilibrated and frozen thawed (37 degrees C for 30s) semen was evaluated for motility, viability and acrosomal integrity, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides these tests, LPO level was assessed in sperm and seminal plasma in equilibrated and frozen thawed semen. Results revealed that addition of oviductal proteins to semen before freezing convey beneficial effect in terms of spermatozoan motility, viability and acrosomal integrity. Nonluteal oviductal proteins favored significantly (P < 0.05) higher sperm penetration distance in cervical mucus (23.00+/-1.15 mm) than the control group (15.00+/-3.46 mm) in frozen thawed semen. Similarly, swollen sperm percentage was also significantly (P < 0.05) higher in NLOP treated group than the LOP included and control groups. In frozen thawed spermatozoa, the LPO level was significantly (P < 0.05) lower in NLOP added group than the LOP added and control group. It was inferred that incorporation of oviductal proteins in extender before freezing reduced the lipid peroxidation levels in buffalo spermatozoa during cryopreservation and thereby improved the post-thaw semen quality.     

Docosahexaenoic acid in diluent for goat semen cryopreservation

The effects of docosahexaenoic acid (DHA) in the diluent for cryopreservation of goat semen on seminal quality and the optimal levels to be used were evaluated. After collection, semen was pooled and physically evaluated, then divided into four aliquots with different DHA levels in the diluent: 0, 10, 20, and 30 ng mL^-1. The semen was cryopreserved in a TK 3000® freezing machine and then thawed for assessment at 37 °C. Sperm motility and vigor, membrane integrity, acrosomal integrity, mitochondrial activity, and sperm chromatin compaction were evaluated after thawing. A completely randomized design was used. For normally distributed variables, ANOVA and regression analysis were used to test for differences between treatments, and for non-parametric data, the Kruskal Wallis test was used at the 5% significance level. There were no differences among groups in terms of membrane integrity, acrosomal integrity, or chromatin compaction. There was a decrease in class I mitochondrial activity with increasing DHA level (P<0.05), but no differences in classes II, III, and IV (P>0.05). The inclusion of 10 to 30 ng mL^-1 of DHA in the diluent did not result in improvements in seminal quality parameters after thawing, with some impairment observed in the mitochondrial activity of the sperm cells.     

Changes in the structures of motile sperm subpopulations in dog spermatozoa after both cryopreservation and centrifugation on PureSperm(®) gradient

The aims of the present study were to: (1) determine if discrete motile sperm subpopulations exist and their incidence in fresh dog ejaculates, (2) evaluate the effects of cryopreservation on the distribution of spermatozoa within the different subpopulations, and (3) determine the effect of the discontinuous PureSperm(?) gradient on the sperm subpopulation structure of frozen-thawed dog spermatozoa. Semen from 5 dogs were collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm(?). Sperm motility (assessed by computerized-assisted semen analysis, CASA) was assessed before freezing, just after thawing and after preparation on the PureSperm(?) gradients. Cryopreservation had a significant (P<0.001) effect on CASA-derived parameters. PureSperm(?) centrifugation yielded sperm suspensions with improved motility (P<0.01). A multivariate clustering procedure separated 19414 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (20.97%), Subpopulation 2 consisting of slow and low-linear spermatozoa (18.24%), Subpopulation 3 consisting of highly active but non-progressive spermatozoa (20.75%), and Subpopulation 4 consisting of high speed and progressive spermatozoa (40.03%). Although, cryopreservation had a significant (P<0.001) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after freezing and thawing. The selected sperm samples was enrich in Subpopulation 4, reaching a proportion of 31.9% of the present spermatozoa, in contrast with the unselected sperm samples, where this sperm subpopulation accounted for 24.9% of the total. From these results, we concluded that four well-defined motile sperm subpopulations were present either in fresh semen, in unselected sperm samples or in selected preparations from dogs. The discontinuous PureSperm(?) gradient is a simple method to improve the quality of canine frozen-thawed semen samples, since Subpopulation 4 (high-speed and progressive spermatozoa) was more frequently observed after preparation on the gradient. Finally, this study also demonstrated that the general motile sperm structure present in dog remains constant despite the effect caused by either cryopreservation or separation on PureSperm(?) gradient.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

Knockout serum replacement is an efficient serum substitute for cryopreservation of human spermatozoa

The freeze-thaw procedure causes irreversible structural and functional changes in human spermatozoa. In order to decrease the detrimental effects of cryopreservation and improve the quality of post-thawed spermatozoa, the constituents of the freezing solution attracted considerable attention. In this study, for the first time, we evaluated the efficacy of knockout serum replacement (KSR) as a substitute for human serum albumin (HSA) for cryopreservation of human spermatozoa. Twenty semen samples were collected from normozoospermic men and divided them into five equal groups. One of the aliquots was diluted with glycerol-based medium as a control group (CON). The other four aliquots were diluted with the sucrose solution containing 5% HSA (H5), 10% HSA (H10), 5% KSR (K5), and 10% KSR (K10). The diluted samples were frozen and preserved in liquid nitrogen. Post thawed sperm parameters including motion characteristics, viability, membrane integrity, mitochondrial activity, acrosome integrity and DNA intactness in all of the sucrose-based groups were comparable with glycerol-based medium. The replacement of HSA by 10% KSR in the freezing medium resulted in significantly higher post-thawed viability, acrosome integrity and DNA intactness compared with other sucrose-based groups. In conclusion, the addition of 10% KSR to the sucrose-based freezing solution improves the quality of post-thawed human spermatozoa and may have potential to develop chemically defined freezing medium.     

DNA fragmentation in chicken spermatozoa during cryopreservation

Semen cryopreservation is fundamental both for the practice of artificial insemination, and for the conservation of genetic resources in cryobanks; nevertheless, there is still not an efficient standard freezing procedure assuring a steady and suitable level of fertility in fowl, and consequently there is no systematic use of frozen semen in the poultry industry. This study examined changes in motility (CASA), cell membrane integrity (Ethidium Bromide (EtBr) exclusion procedure and stress test) and DNA fragmentation (neutral comet assay) in fowl spermatozoa before, during and after cryopreservation and storage at −196 °C. An optimized comet assay for chicken semen was studied and applied to the analyses. Semen collected from 18 Mericanel della Brianza (local Italian breed) male chicken breeders was frozen in pellets and thawed in a water bath at 60 °C. Measurements were performed on fresh semen soon after dilution, after equilibration with 6% dimethylacetamide at 4 °C (processed semen) and after thawing. Sperm DNA damage occurred during cryopreservation of chicken semen and the proportion of spermatozoa with damaged DNA significantly increased from 6.2% in fresh and 6.4% in processed semen to 19.8% in frozen-thawed semen. The proportion of DNA in the comet tail of damaged spermatozoa was also significantly affected by cryopreservation, with an increase found from fresh (26.3%) to frozen-thawed (30.9%) sperm, whereas processed semen (30.1%) didn't show significant differences. The proportion of total membrane damaged spermatozoa (EtBr exclusion procedure) did not increase by 4 °C equilibration time, and greatly and significantly increased by cryopreservation; the values recorded in fresh, processed and frozen semen were 2.9, 5.6, and 66.7% respectively. As regards the proportion of damaged cells in the stress test, all values differed significantly (7.1% fresh semen, 11.7% processed semen, 63.7% frozen semen). Total motility was not affected by equilibration (52.1% fresh semen, 51.9% processed semen), whereas it decreased significantly after cryopreservation (19.8%). These results suggest a low sensitivity of frozen-thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation.     

Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate

Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10 mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca²⁺]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P < 0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca²⁺]i between P1 and SRF-P1 in fresh as well as in frozen–thawed semen. A higher (P < 0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32 kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.     

A differential mechanism is involved during heparin- and cryopreservation-induced capacitation of bovine spermatozoa

After ejaculation, mammalian spermatozoa must undergo capacitation to fertilize. Capacitation of bovine spermatozoa occurs in vitro in medium supplemented with heparin. Semen cryopreservation is an important tool for assisted reproduction, although the fertility of frozen-thawed spermatozoa is reduced, possibly due to precocious capacitation-like changes that are known to occur. Our purpose was to clarify the mechanisms involved in bull sperm cryocapacitation induced by cryopreservation. Our general hypothesis is that the signaling pathways that lead to capacitation are triggered by the cryopreservation procedure. Ejaculated bovine semen was divided into two aliquots and diluted in extender; one was then kept fresh, whereas the second was cryopreserved. Western blots of extracted sperm proteins with anti-phosphotyrosine antibody showed that capacitation, induced by either heparin in fresh sperm or cryopreservation (cryocapacitation), is associated with a differential profile of phosphotyrosine-containing proteins. Immunolocalization of phosphotyrosine-containing proteins in the fresh and cryopreserved spermatozoa showed that, after thawing, cryocapacitated sperm displayed labeling over the acrosomal region, whereas for fresh sperm, this labeling appeared after 5-h incubation with heparin. The chlortetracycline assay and the ability of the sperm to undergo the lysophosphatidylcholine-induced acrosome reaction were used to confirm that a subpopulation of cryopreserved sperm is capacitated at thawing, irrespective of heparin inclusion. Since glucose is known to inhibit heparin-induced capacitation, the semen extender was modified to include glucose as a means of inhibiting cryocapacitation; however, cryocapacitation was not prevented according to the chlortetracycline assay and profile of phosphotyrosine-containing sperm proteins.     

Effect of semen collection method (artificial vagina vs. electroejaculation), extender and centrifugation on post-thaw sperm quality of Blanca-Celtibérica buck ejaculates

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl(?), Andromed(?) and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1-/PI- (intact spermatozoa), and Mitotracker+/YO-PRO-1- (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl(?) and Andromed(?) resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl(?) or Andromed(?) are used as freezing extenders.     

Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles

This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract. Blastocyst development after ICSI was equivalent between control spermatozoa and motile 2F spermatozoa (27 and 23%, respectively). Blastocyst development after injection of non-motile 2F spermatozoa (13%) tended (P=0.07) to be lower than that for control spermatozoa. Injection of sperm extract into oocytes that received non-motile 2F spermatozoa resulted in a significant decrease in blastocyst development (to 2%) compared with injection of non-motile 2F spermatozoa alone. Spermatozoa from a subfertile stallion was similarly processed and used for ICSI; blastocyst development for both motile control (once frozen) spermatozoa and motile 2F spermatozoa was 9%. In conclusion, frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI. Non-motile spermatozoa from reprocessed semen may also achieve embryo development after ICSI. To our knowledge, this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species.     

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 degrees 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 degrees C and stored for 1h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 degrees 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 1h of incubation at 37 degrees C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6h 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 6h 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.     

Measurement of the sperm motility index via the sperm quality analyzer and its relationship to other qualitative sperm parameters

Sperm parameters such as the concentration and percentage of motile spermatozoa are commonly used to assess semen quality. The sperm quality analyzer (SQA) is a device that detects variations in the optical density of motile spermatozoa, providing a sperm motility index (SMI) that is based on various sperm parameters including the concentration, morphology and acrosomal status of motile spermatozoa. The relationship between SMI values of frozen-thawed bovine spermatozoa undergoing swelling in a hypoosmotic medium (100 mOsm/L) and other sperm parameters were evaluated. Frozen semen specimens from 3 bulls were thawed and washed with Ham's F-10 supplemented with 3% BSA and split into 3 (0.2 mL) aliquots. The aliquots were diluted with 1.0 mL of Ham's F-10 (Aliquot 1), isotonic sodium citrate (Aliquot 2), and hypotonic sodium citrate (Aliquot 3). The osmotic pressure of the media used for dilution of Aliquots 1 and 2 was 300 mOsm/L, while that for Aliquot 3 was 100 mOsm/L. Following dilution, the aliquots were incubated for 30 min and manually assessed at 5-min intervals for the percentage and grade of motility (Grades 0 to 4) as well as for the percentage of swollen spermatozoa. Sperm samples were simultaneously evaluated by SQA to obtain the SMI values at the same 5-min intervals during the 30-min incubation. Significant correlations were observed between SMI values and other sperm parameters in Aliquot 3 (P < 0.05). The results indicated that the SMI values obtained from frozen-thawed bovine spermatozoa exposed to a 100 mOsm/L diluent, which causes optimal swelling of spermatozoa, are highly correlated to other sperm parameters. The SQA unit, as applied in this study, can be used for rapid and reliable screening of sperm samples.