Effect of filtration on post-thaw quality of bull semen

Semen from 4 Holstein bulls was diluted in 4 different extenders, filtered with Sephadex ion-exchange column, and frozen in liquid nitrogen. Sperm motility, progressive motility, path velocity, progressive velocity and the percentage of normal acrosomes of filtered and nonfiltered semen were recorded before and after freezing. Semen characteristics were significantly influenced by extender, filtration and freezing. Before and after freezing, motility measurements and the percentage of normal acrosomes were higher (P < 0.001) in filtered than in nonfiltered spermatozoa. Post-thaw recovery rate of motile spermatozoa was higher in filtered semen than nonfiltered (68 vs 39%, P < 0.0001). The reduction in motility, progressive motility and the percentage of normal acrosomes during freezing and thawing processes were significantly lower (P < 0.0001) in filtered semen (34, 34 and 4%, respectively) than nonfiltered (59, 54 and 15%, respectively). Post-thaw viability of spermatozoa was significantly affected by extender, filtration and time (P < 0.0001). Immediate (0 h) post-thaw motility of nonfiltered semen (29%) was similar to 4-h post-thaw motility of filtered semen (25%; P > 0.05). In conclusion, bull spermatozoa recovered by Sephadex ion-exchange filtration showed better post-thaw viability.     

The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa

Cryopreservation of epididymal spermatozoa is a potentially valuable tool for preserving genetic material from individuals of endangered species that die accidentally. Improvement of sperm-freezing protocols would increase the efficacy of gene banking from endangered felids, and the domestic cat can be used as a model for the wild felids. Addition of the detergent Equex STM paste to semen freezing extenders has been found to improve post-thaw survival and longevity of spermatozoa from various species but has never been tested for cat spermatozoa. Spermatozoa from cats with a high percentage of morphologically abnormal spermatozoa are more susceptible for cold injury and osmotic stress than spermatozoa from normozoospermic cats. Therefore, the aims of this study were to investigate: (a) if addition of Equex STM paste to a semen freezing extender would improve post-thaw sperm survival, and (b) if there is a relation between the percentage of morphologically normal spermatozoa and cryopreservation induced damage in cat epididymal spermatozoa. Spermatozoa harvested from epididymides of 10 male cats were frozen in a Tris egg yolk extender with or without the addition of Equex STM paste (0.5%, v/v). Sperm motility, membrane integrity and acrosomal status were evaluated immediately after harvesting, and at 0, 2, 4 and 6 h post-thaw. Sperm membrane integrity and acrosomal status were also evaluated after cooling to 4 degrees C, just before freezing. Cooling did not cause significant damage to the spermatozoa, whereas freezing damaged sperm membranes and acrosomes. Addition of Equex to the freezing extender had a significant positive effect on the percentage of intact acrosomes immediately after thawing (P > 0.05), but had a negative effect on the longevity of the spermatozoa; the percentages of membrane intact and motile spermatozoa being significantly lower in the presence of Equex than in the controls at 6h after thawing. The percentage of morphologically normal spermatozoa was not found to be correlated with either cryopreservation induced acrosome or plasma membrane damage, or with post-thaw motility (P > 0.05). The results clearly show that addition of Equex STM paste in the freezing extender protects the acrosomes of cat epididymal spermatozoa during the freezing--thawing process, but reduces the sperm longevity during in vitro incubation at 38 degrees C. Our results also indicate that the percentage of morphologically normal epididymal spermatozoa is not correlated with cryopreservation induced sperm damage using the described freezing protocol.     

The effect of glycerol-related osmotic changes on post-thaw motility and acrosomal integrity of ram spermatozoa

Ram semen, collected by artificial vagina, was diluted and processed for long-term storage as described by P. S. Fiser, L. Ainsworth, and R. W. Fairfull (Canad. J. Anim. Sci. 62, 425-428, 1982). The concentration of the cryoprotectant, glycerol, was adjusted to 4% in the diluted semen prior to freezing by a one-step addition at 30 degrees C (Method 1), by cooling the semen to 5 degrees C and addition of the glycerol gradually over 30 min (Method 2), by one-step addition of glycerol prior to equilibration for 2 hr (Method 3), or by cooling to 5 degrees C, followed by a holding period of 2 hr at 5 degrees C, and the one-step addition of glycerol just prior to freezing (Method 4). After thawing, the glycerol concentration of the semen was reduced by stepwise dilution from 4 to 0.4% over 15 or 30 min or by a one-step ten-fold dilution. The average post-thaw percentage of motile spermatozoa was significantly lower after addition of glycerol by Method 1 (39.9%) than when the glycerol was added by the other three methods (range, 44.0-46.4% averaged over the glycerol dilution). The average post-thaw percentage of intact acrosomes (61.2%), highest in semen in which the glycerol was added by Method 2, was not significantly different from those in which glycerol was added to semen by Methods 3 and 4, but it was significantly higher than that found in semen in which the glycerol was added by Method 1 (54.4%). However, when averaged over the method of glycerolation, the post-thaw percentage of motile spermatozoa (range, 43.7-44.2%) and the percentage of intact acrosomes (range, 56.8-59.5%) did not differ significantly in semen subjected to gradual decrease in glycerol concentration and diluent osmolality (over 15 and 30 min) or by a one-step, 10-fold dilution. These data indicate that post-thaw survival of spermatozoa can be influenced by the way in which glycerol is added prior to freezing. However, post-thaw spermatozoa motility and acrosomal integrity can be maintained even after a rapid decrease in glycerol concentration such as that which accompanies insemination or dilution of semen for assessment of motility.     

Comparison of different techniques to determine the percentage of intact acrosomes in frozen-thawed bull semen

Twenty-seven different batch dates of frozen bull semen from 26 bulls were used in this study. The semen was in 0.5-ml straws, and 23 of the batch dates were in whole milk extender while 4 were in egg yolk-tris extender. Straws from each batch of semen were incubated for 2 hours in a water bath at 37 degrees C. Following this, the percentage of progressive motility, the rate of motility, and the percentage of intact acrosomes were determined for each unfixed sample. Each batch of semen was fixed in 2 different solutions of 0.2% glutaraldehyde in phosphate-buffered saline (glutaraldehyde 1 and glutaraldehyde 2) and in 10% neutral buffered formol saline (formol saline). The percentage of intact acrosomes for each sample in these fixatives was determined at Day 0 and Day 7. There were no significant differences in the percentages of intact acrosomes among the unfixed samples and the samples in the 3 fixatives at Day 0. At Day 7, the samples in formol saline had a significantly higher percentage of intact acrosomes than those in glutaraldehyde 2. When the percentage of intact acrosomes for the unfixed samples at Day 0 was compared with the percentages of intact acrosomes for glutaraldehyde 1, glutaraldehyde 2, and formol saline at Day 7, only the percentage of intact acrosomes for formol saline was significantly higher than for the unfixed samples. Only one of the batches of semen in egg yolk-tris extender could be evaluated in formol saline because of a heavy precipitate that formed. There was a significant interaction between extender and storage. For the whole milk extender, the percentages of intact acrosomes at Day 7 were higher than for Day 0 for all the fixatives used. For the egg yolk-tris extender, the percentage of intact acrosomes decreased from Day 0 to Day 7. The correlations between the percentage of intact acrosomes for the unfixed samples and the post-incubation percentage of progressivé motility and rate of motility were 0.65 and 0.46, respectively.     

Effect of post-thaw incubation on sperm kinematics and acrosomal integrity of ram spermatozoa cryopreserved in medium-sized French straws

The objectives were to assess the effect of post-thaw in vitro incubation on motion characteristics and acrosomal integrity of ram spermatozoa of native Malpura and Bharat Merino breeds maintained under a semi-arid tropical environment. Good quality semen samples of both breeds were diluted, packaged in medium-sized straws, and frozen under controlled conditions. Straws were thawed at 60 degrees C for 10s and thawed samples were incubated at 37 degrees C for 4h. Post-thaw motion characteristics and acrosomal integrity of incubated spermatozoa were assessed (by computer-aided semen analysis and Giemsa staining, respectively) just prior to incubation and at hourly intervals thereafter. There was a significant effect of incubation time on motility characteristics and the proportion of spermatozoa with normal acrosomes; 81.4% (arcsin transformed value, 65.2) of spermatozoa were motile at the start of incubation, with 47.9% (arcsin transformed value, 44.4) motile after 4h. At the corresponding times, there were normal acrosomes in 65.8 (arcsin transformed value, 54.8) and 55.7% (arcsin transformed value, 48.9) of spermatozoa, respectively. The percentage straightness of spermatozoa varied during incubation (P < 0.01). However, there was no significant change in percentage linearity, curvilinear velocity, average path velocity, straight line velocity, lateral head displacement, and beat cross frequency of spermatozoa during incubation. There were no breed variations in any motility parameters during incubation, except percentage straightness (P < 0.05), lateral head displacement (P < 0.05) and beat cross frequency (P < 0.01). That sperm motility and acrosomal morphology were very acceptable immediately post-thaw and after 4h of incubation indicated the efficacy of cryopreserving ram spermatozoa under controlled conditions in medium-sized straws.     

Effect of glycerol concentration on frozen boar sperm

In experiment 1, Beltsville F3 extender containing 0 to 7% glycerol was added to boar sperm. Glycerol was either retained during freezing or removed by centrifugation before freezing. When glycerol was retained, there was a significant negative linear relationship between the percentage of sperm acrosomes with a normal apical ridge (NAR) and the percentage of glycerol. When glycerol was removed before freezing, the percentage of NAR acrosomes did not differ among samples. The percentage of motile sperm and the percentage of glycerol in the original extender were linearly related regardless of whether glycerol was retained or removed before freezing.In experiment 2, four concentrations of glycerol, three cooling times and two dilution rates were compared when semen was frozen in Beltsville F5 extender. The post-thaw results for percentages of NAR acrosomes and sperm motility were optimum with 1% glycerol and a 1:4 dilution rate. Cooling time had a minor effect on the freezing results.In experiment 3, the competitive fertilizing capacity of boar sperm frozen with 1% glycerol was compared with that frozen without glycerol. The number of ova fertilized by sperm frozen with 0 or 1% glycerol, 52 and 72 ova, respectively, were nearing statistical difference from a 50:50 ratio (P<.07). These results indicated that under the conditions of this study glycerol was of some positive value as a cryoprotectant for boar sperm.     

Transitory acidification of semen as a potential method for the inactivation of some pathogenic microorganisms. Effect on fertilization and development of ova in superovulated heifers

The fertilizing capacity of transitorily acidified semen was investigated with respect to using acidification as a method for destroying or inactivating acid labile pathogenic microorganisms in semen prior to freezing. Ejaculates diluted 1:1 with phosphate buffered saline (PBS) were acidified to pH 5.0 for 2 or 5 minutes before being returned to their original pH. They were then frozen by commercial methods and used to artificially inseminate superovulated heifers. A total 739 ova and embryos was collected from 119 inseminated donors. The fertilization rate was 88%, and 78% of the embryos were of transferable quality. Semen acidification had no apparent effect on the post-thaw percentage of intact acrosomes.     

Ultrastructural and cytochemical characterization of follicular cell types in bovine (Bos taurus) cumulus-oocyte complexes aspirated from small and medium antral follicles during the estrus cycle

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.     

The effect of straw size, freezing rate and thawing rate upon post-thaw quality of dog semen

Optimal freeze-thaw processes for dog semen will yield a maximal number of insemination doses from an ejaculate. The objectives of this study were to compare the effects of two straw sizes (0.25- and 0.5-mL French), two freezing rates (straws suspended 3.5 and 8 cm above liquid nitrogen) and two thawing rates (in water at 37 and 70 degrees C) upon post-thaw quality of dog semen, and to determine the best treatment combination. Quality was expressed in terms of the percentage progressively motile sperm 5 and 60 min after thawing and the percentage of abnormal acrosomes 5 min after thawing. One ejaculate from each of eight dogs was frozen. Two straws from each ejaculate were exposed to each of the eight treatment combinations. Data were analyzed by means of a repeated measures factorial analysis of variance and means compared using Bonferroni's test. Dog affected each response variable (P < 0.01). Neither straw size, nor freezing rate, nor thawing rate affected motility 5 min after thawing (P > 0.05). Half-milliliter straws resulted in 5.7% more progressively motile sperm 60 min after thawing and 6.5% fewer abnormal acrosomes than 0.25-mL straws (P < 0.05, n = 64). The percentage progressively motile sperm 60 min after thawing tended to be higher for semen thawed at 70 degrees C compared to 37 degrees C (P < 0.06, n = 64). Semen thawed in water at 70 degrees C had 6.6% fewer abnormal acrosomes than semen thawed in water at 37 degrees C (P < 0.05, n = 64). Freezing rate interacted with thawing rate (P < 0.05) in their effects upon acrosomal morphology and freezing 8 cm above liquid nitrogen and thawing in water at 70 degrees C was best. Dog semen should be frozen in 0.5-mL straws, 8 cm above liquid nitrogen and thawed in water at 70 degrees C.     

Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa

We investigated the use of duck egg yolk (DEY), Guinea fowl egg yolk (GFEY) and Indian indigenous hen (Desi) egg yolk (IDEY) in extender for improving the post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa, and compared it with commercial hen egg yolk (CHEY; control). For this purpose, two consecutive ejaculates of semen from each of two Nili-Ravi buffalo bulls were collected on 1 day each week for 5 weeks (replicates; n=5) with artificial vagina (42 degrees C). Split pooled ejaculates, were diluted in tris-citric acid glycerol extender containing either DEY or GFEY or IDEY or CHEY at 37 degrees C. Extended semen was cooled to 4 degrees C in 2 h and equilibrated for 4 h at 4 degrees C. Cooled semen was then filled in 0.5 ml straws at 4 degrees C and frozen in programmable cell freezer. Thawing of semen was performed at 37 degrees C for 30 s. Sperm motility, plasma membrane integrity and sperm morphology (acrosome integrity, head, mid-piece and tail abnormalities) of each semen sample were assessed at 0, 3 and 6 h after thawing and incubation at 37 degrees C. Visual motility (%) and percentage of intact plasma membranes assessed at 6h post-thaw of buffalo bull spermatozoa were highest (P<0.05) due to DEY as compared to GFEY, IDEY and control. The percentage of spermatozoa with normal acrosomes at 0, 3 and 6 h post-thaw was highest (P<0.05) in DEY extender than GFEY, IDEY and CHEY. Sperm tail abnormalities (%) observed at 0, 3 and 6 h post-thaw in samples cryopreserved with freezing extender having DEY were lower (P<0.05) as compared to extender containing GFEY, IDEY and CHEY. In conclusion, DEY compared to other avian yolks in extender improves the frozen-thawed quality of buffalo bull spermatozoa.     

Effects of different cryoprotective agents on ram sperm morphology and DNAintegrity

This study investigates the effects of glycerol, 1,2 propanediol, sucrose, and trehalose on post-thaw motility, morphology, and genome integrity of Awassi ram semen. Ejaculates of thick consistency with rapid wave motion (>+++) and >70% initial motility were pooled. Sperm were diluted to a final concentration of 1/5 (semen/extender) in 0% cryoprotectant, 6% glycerol, 6% 1,2 propanediol, 62.5 mM sucrose or 62.5 mM trehalose using a two-step dilution method. The equilibrated semen was frozen in 0.25-ml straws. Semen samples were examined for sperm motility, defective acrosomes (FITC-Pisum sativum agglutinin (FITC PSA)), DNA integrity (acridine orange staining (AO)) and apoptotic activity (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Caspase-3 activity) at four time points: after dilution with extender A, after cooling to 5 °C, after equilibration and post-thaw. Freezing and thawing procedures (cooling at 5 °C, dilution, equilibration, and thawing) had negative effects on motility (P < 0.001), acrosome integrity (P < 0.001), and DNA integrity as determined by AO (P < 0.001) and TUNEL (P < 0.001) assays. There were positive correlations between sperm with defective acrosomes and apoptotic (AO- and TUNEL-positive) spermatozoa. In contrast, a significant negative correlation was found between sperm motility and defective acrosomes and AO- and TUNEL positivity (P < 0.01). The cryopreservation process acts as an apoptotic inducer in ram semen; all cryoprotectants used in the present study allowed apoptosis to some extent, with negative effects on sperm morphology and DNA integrity. The glycerol group performed better than the propanediol, sucrose, trehalose, and control groups in terms of post-thaw sperm motility but not DNA integrity.     

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 of Equex STM Paste on the quality of frozen-thawed epididymal dog spermatozoa

This study was carried out to investigate the cryoprotective efficacy of Equex STM Paste on the quality of canine post-thaw epididymal spermatozoa. Following castration, spermatozoa were flushed from the cauda epididymides. Epididymal spermatozoa from 13 of 16 dogs with a sperm motility of >70% were frozen in an egg yolk-Tris extender, supplemented with Equex STM Paste (0.5%, v/v); the extender free of Equex STM Paste served as a control cryoprotective diluent. The quality of spermatozoa, judged by its motility, plasma membrane integrity and acrosome integrity, was evaluated on four occasions, immediately after collection, after equilibration and at 0 and 2h post-thaw. Reducing the temperature to 4 degrees C for 2h prior to freezing decreased sperm motility (P=0.001), but had no effects on membrane integrity or acrosome integrity. Immediately after thawing, the percentage of acrosome-intact spermatozoa significantly decreased in samples frozen without Equex STM Paste compared to freshly collected or Equex-treated samples. After incubation at 37 degrees C for 2h post-thaw, a greater percentage of motile spermatozoa (P=0.018) and spermatozoa with intact acrosomes (P=0.001) were observed in Equex-treated samples compared with the control. The percentage of membrane-intact spermatozoa did not differ significantly between Equex-treated and control samples at any time. Supplementation with Equex STM Paste in the semen extender was effective for freezing canine epididymal spermatozoa because it protected acrosome integrity against damage induced by cryopreservation and it prolonged post-thaw sperm motility during in vitro incubation at 37 degrees C.     

Effect of post-thaw dilution with autologous prostatic fluid on dog semen motility and sperm acrosome status

The aim of this study was to evaluate the effect of post-thaw dilution with autologous prostatic fluid on motility parameters, longevity and acrosome status of frozen-thawed dog spermatozoa. After semen collection, seminal plasma was separated by centrifugation and stored frozen until use. Sperm pellets were diluted in two steps with an egg yolk-Tris extender to a final concentration of 5% glycerol and 0.5% Equex STM Paste. After thawing, semen was diluted 1:2 either with Tris buffer or with the autologous prostatic fluid. Motility was evaluated using a phase contrast microscope and a computer-assisted motility analyser system immediately after thawing and at hourly intervals up for 4h at 38 degrees C. The status of acrosomes was assessed with Spermac stain at thawing and after 2 h of incubation. Motility and straight line velocity were initially higher in prostatic fluid-diluted samples (0 h and 0 and 1h, respectively), but decreased to values similar to those of Tris-diluted samples in a time-dependent manner. In contrast, both the curvilinear velocity and amplitude of lateral head displacement were lower in prostatic fluid-diluted samples (1 and 3 h and 0, 1 and 3 h, respectively). The dilution did not have any significant effect on the percentage of acrosome-intact spermatozoa at either thawing or after 2 h. The pattern of motility of prostatic fluid-diluted samples suggests a reduction in hyperactivated motility with time, even though prostatic fluid neither prolonged spermatozoa longevity nor had any effect on the status of spermatozoa acrosomes.     

Effect of glutaraldehyde concentration and fixative temperature on the number of spermatozoa with normal acrosomes in goat semen

Two experiments were conducted to determine the best fixative solution and the most suitable temperature for fixing sperm cells from goat ejaculates. In Experiment 1, a 6x3 factorial design was used to test 4 glutaraldehyde concentrations (0.5, 1, 2 and 4%) plus 1 treatment that did not contain a fixative (0%) but to which 0.3% sodium fluoride (NaF) had been added to immobilize the spermatozoa; the control treatment contained no fixative or NaF. The 6 treatments were tested with 3 different solvents: PBS, Na citrate and BL-1, representing a total of 18 samples per replicate. The fixed samples always provided a significantly higher (P<0.01) percentage of normal acrosomes than the unfixed samples, whether immobilized with NaF or observed inmediately after dilution. In Experiment 2, a 3x3 factorial design was used to determine the effect of the temperature of the glutaraldehyde fixative solution on the number of morphologically normal acrosomes from goat semen samples kept at 3 different temperatures. Our findings indicated that at all 3 fixative solution temperatures (5, 20 and 37 degrees C) there was a significant difference (P<0.01) in the percentage of normal acrosomes. At 5 degrees C, glutarhaldehyde yielded a general mean number of 53.6 normal acrosomes vs 75.1 at 20 degrees C and 83.05 at 37 degrees C. Based on these results, we recommend that the temperature of a fixative solution be established when designing an experiment using goat semen, since the temperature has a significant effect on the number of the normal acrosomes found in a semen sample.     

Centrifugation on PureSperm(®) density-gradient improved quality of spermatozoa from frozen-thawed dog semen

The main objective of this study was to investigate if centrifugation through PureSperm^® density-gradient can improve the post-thaw semen quality of dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm^®. Assessments of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of Propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, unselected samples and selected preparations. Cryopreservation had a significant (P < 0.001) effect on all studied semen parameters. PureSperm^® centrifugation yielded sperm suspensions with improved motility and viability (P < 0.001). The washing step significantly reduced (P < 0.001) all of the kinematics parameters evaluated as well as reduced the proportion of viable spermatozoa with intact acrosomes (P < 0.05). We concluded that PureSperm^® centrifugation is a successful method for improving the quality of frozen-thawed dog spermatozoa. However, washing after density-gradient centrifugation dramatically reduces the post-thaw semen quality, indicating that the inclusion of such a washing step is unnecessary.     

The effect of dialysis of extended ram semen prior to freezing on post-thaw survival and fertility

The effect of dialysis on extended ram semen prior to cryopreservation was studied. Techniques were developed to improve post-thaw recovery of dialyzed semen and a fertility trial was used to evaluate the viability of dialyzed and frozen semen. Dialysis prior to freezing was shown to increase post-thaw recovery of motile cells and percentage of cells passing through a Sephadex filter. Freezing semen in pellets on dry ice was superior to freezing in French straws. Pellets were thawed in an aluminum thaw block at 42 to 45 degrees C before insemination of progestagen-PMSG synchronized ewes. Double inseminations were made at 12-hr intervals. Natural service of synchronized ewes was also made at 12-hr intervals as a control. There was no significant difference (P greater than 0.05) in fertility between naturally serviced ewes (44.4%) and ewes inseminated with frozen semen (44.7%).     

Effect of sericin supplementation on heat shock protein 70 (HSP70) expression,redox status and post thaw semen quality in goat

Cryopreservation results in substantial deterioration of heat shock protein 70 (HSP70) and ultra-structural changes in sperm organelles, resulting in a marked reduction in post-thaw semen quality. The present study was aimed to explicate the effect of sericin supplementation on expression profile of HSP70, redox status and post-thaw semen quality in Barbari goat. Five Barbari bucks were used to collect thirty semen ejaculates by using artificial vagina and each ejaculate was divided into three aliquots to which sericin was supplemented at 0% (Control), 0.25% (T1) and 0.50% (T2). Further, extended semen samples were equilibrated followed by their cryopreservation. Post-thaw semen characteristics, redox status of seminal plasma, enzyme leakage and HSP70 gene/protein expression in spermatozoa were assessed in all the groups. Per cent progressive motile spermatozoa, spermatozoa having intact plasma membrane (HOST + ve) and intact acrosomes in post-thaw spermatozoa were significantly (p < 0.01) higher in T1 and T2 as compared to control. A significant (p < 0.01) reduction in abnormal spermatozoa was found in T1 as compared to T2. Sericin supplementation significantly (p < 0.05) improved the antioxidative status (SOD, GST, CAT), reduced lipid peroxidation (MDA) and also prevented enzyme (ALT, LDH) leakage as compared to control samples. qRT-PCR results revealed that HSP70 mRNA expression was significantly (p < 0.01) upregulated in T1 and T2 group as compared to control. The positive effect of sericin on expression of HSP70 was further confirmed by immunoblotting followed by densitometry revealing higher expression in T1 and T2 compared to control. Inclusion of 0.25% w/v sericin in semen extender ameliorated the post-thaw semen quality by improving antioxidative status and minimizing the leakage of intracellular enzymes. Sericin supplementation had a beneficial effect on HSP70/HSP70 mRNA expression either by induction or by protection of HSP70/HSP70 mRNA as evident from the gene expression and immunoblotting studies.     

Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility.