Evaluation of fresh and frozen-thawed fowl semen by flow cytometry

The aim of this study was to assess the spermatozoal viability, acrosome integrity, mitochondrial activity, and DNA status in the frozen-thawed fowl semen with the use of flow cytometry. The experiment was carried out on 10 sexually adult roosters of meat type line Flex. The semen was collected three times a week by dorso-abdominal massage method, then pooled and subjected to cryopreservation using “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. For cytometric analysis the fresh and frozen-thawed semen was extended with EK diluent to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI). Acrosomal damages were evaluated using phycoerythrin-conjugated lectin PNA from Arachis hypogaea. The percentage of live spermatozoa with functional mitochondria was estimated using Rhodamine 123 (R123) and PI. The spermatozoal DNA integrity was measured by sperm chromatin structure assay (SCSA). The freezing-thawing process decreased the viability, mitochondrial activity in the chicken sperm and increased the percentage of dead cells with ruptured and intact acrosomes, and also the percentage of spermatozoa with fragmented DNA. In conclusion, the present study indicates that fluorescent staining and flow cytometry may be useful for assessment of the changes of fowl semen quality caused by cryopreservation process. This technique allows precise examination of spermatozoa functional characteristic in a very short time.     

Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic cod, Gadus morhua L

There is a lack of biomarkers or indices that can be used to predict the quality of fish semen samples following the freezing and thawing cycle. In the present study, a series of semen indices were tested to assess if they could accurately forecast the cryopreservation potential of Atlantic cod (Gadus morhua) semen. Fresh and frozen-thawed sperm activity variables were compared, and relationships between frozen-thawed sperm activity and fertilization success were examined. In comparison with fresh sperm, activity variables of frozen-thawed spermatozoa were reduced. Of the 18 males examined, mean (± SEM) spermatocrit of fresh sperm was 40.72 ± 4.23%, osmolality of the seminal plasma 366.32 ± 4.95 mOsmol/kg, pH 8.32 ± 0.04, protein concentration 1.05 ± 0.08 mg/mL, anti-trypsin activity 153.83 ± 19.25 U/L, and total antioxidant capacity 0.15 ± 0.03 μmol Trolox equivalents/mL. Frozen-thawed fertilization success was highly variable among males with values ranging from 18.5 to 90.2%. Regressions yielded significant positive relationships between frozen-thawed motility, velocity, track crossing frequency, and subsequent fertilization success. Sequential multiple regressions explained up to 95% of the variation in frozen-thawed sperm activity. Spermatocrit and pH of fresh semen were negatively related, whereas osmolality and antioxidant capacity were positively related to frozen-thawed motility and velocity. Each of these indices can be measured within minutes of collecting a fresh sample of semen and are thus early indicators of the capacity of semen samples to withstand cryopreservation. These results have many benefits for conservation of wild stocks, aquaculture production, and for understanding semen biology and cryobiology of fishes.     

Detection of lipid peroxidation in frozen-thawed avian spermatozoa using C(11)-BODIPY(581/591)

The aim of this study was to perform flow cytometric analysis of C11-BODIPY581/591 oxidation in fowl and geese sperm as a marker for membrane lipid peroxidation (LPO) and to establish if the cryopreservation process would make sperm membranes more susceptible to oxidative stress. The experiment was carried out on 10 meat type line Flex roosters and 10 White Koluda® geese. The semen was collected two times a week, by dorso-abdominal massage method and pooled from 10 individuals of each species. Fowl semen samples were subjected to cryopreservation using the “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. Geese semen samples were cryopreserved in plastic straws in a programmable freezing unit with Dimethyloformamide (DMF) as the cryoprotectant. A fluorescent lipid probe C11-BODIPY581/591 provided with two double bonds that are oxidized during their contact with ROS, was used for the purpose of the assessment of the LPO in freshly diluted semen samples and frozen-thawed semen samples. This probe changes its color according to its state (non peroxidized: red; peroxidized: green). Flow cytometric analysis was used to monitor these changes. The White Koluda® geese fresh semen had a higher level of LPO than the Flex fresh semen (P > 0.01). The cryopreservation of fowl semen significantly (P > 0.01) increased the percentage of live and dead spermatozoa with lipid peroxidation. In frozen-thawed semen of White Koluda® geese the percentage of live spermatozoa with LPO significantly decreased (P > 0.05) whereas significantly (P > 0.01) higher level of dead cells with LPO was observed. There were significant differences between the two studied species. After thawing, the percentage of live and dead spermatozoa with lipid peroxidation was higher in fowl semen than in geese semen (P > 0.01). In conclusion, our data clearly indicate the existence of species specific differences in susceptibility of spermatozoa to the oxidation of PUFAs in the cell membranes, where such oxidation is caused by cryopreservation. This study shows that avian spermatozoa are vulnerable to radicals and frozenthawed sperm have higher level of LPO than fresh sperm. According to our observation, fowl semen is more susceptible to LPO than geese semen.     

Use of single-layer centrifugation with Androcoll-C to enhance sperm quality in frozen-thawed dog semen

The aim of this study was to investigate whether single-layer centrifugation (SLC) with Androcoll-C could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from five dogs was collected and cryopreserved following a standard protocol. After thawing, the semen samples were divided in two aliquots, one of which was used as a control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining), viability (dual staining with propidium iodine/acridine orange), and acrosome integrity (dual staining with propidium iodine/isothiocyanate-labeled peanut [Arachis hypogaea] agglutinin) were performed on aliquots of fresh semen, frozen-thawed control samples, and frozen-thawed SLC-treated preparations. A multivariate clustering procedure separated 57,577 motile spermatozoa into three subpopulations (sP): sP1 consisted of poorly active and nonprogressive spermatozoa (48.8%), sP2 consisted of moderately slow but progressive spermatozoa (13.3%), and sP3 consisted of highly active and/or progressive spermatozoa (37.8%). SLC with Androcoll-C yielded sperm suspensions with improved motility, viability, and acrosome integrity (P < 0.01). The frozen-thawed SLC-treated samples were enriched in sP3, representing 38.5% of the sperm population. Likewise, sP2 was more frequently observed after SLC, but not significantly so. From these results, we concluded that for dog semen samples selected by SLC with Androcoll-C after thawing, the sperm quality parameters, including motility patterns, are better than in frozen-thawed control samples.     

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.     

Cryopreservation of bull spermatozoa alters the perinuclear theca

The perinuclear theca (PT) is involved in several important sperm functions leading to fertilization. The objective of this study was to investigate the effect of cryopreservation of bull spermatozoa on the integrity of the PT and the relationship between PT integrity and semen characteristics. Semen from seven bulls was evaluated before and after cryopreservation, comparing the integrity of the plasma membrane (hypo-osmotic test), percentage of live and dead spermatozoa (triple stain), acrosome integrity (triple stain) and the integrity of the PT (negative stain by electron microscopy). Cryopreservation of bull semen caused substantial damage to the PT; the proportion of spermatozoa with a damaged PT was 15.2% versus 52.5% (P<0.05) in fresh versus frozen-thawed spermatozoa, respectively. Furthermore, on average, 67.4% (range, 64-72%) of fresh spermatozoa were live, compared to 53.1% (range, 49-58%) for frozen-thawed spermatozoa; there was an inverse correlation between the percentage of live spermatozoa and the percentage with damage to the PT. Although 59.1% of frozen-thawed spermatozoa had an intact acrosome, only 43.7% of them still remained alive. In frozen-thawed semen, there was a high correlation (r=0.69) between live spermatozoa with an intact acrosome and spermatozoa that maintained an intact PT. In conclusion, freezing/thawing of bull spermatozoa altered the PT and maintaining PT integrity may be necessary to maintain acrosome integrity.     

Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla)

We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa.     

In vitro characteristics of canine spermatozoa subjected to two methods of cryopreservation

The in vitro viability of canine spermatozoa was evaluated after freezing-thawing using the Andersen method, and the commercial CLONE method. These methods differ in the extenders used, number of dilution steps, and equilibration times as well as in both freezing and thawing techniques and rates. Insemination with semen frozen-thawed by either method gives high whelping rates in practice, implying that dog spermatozoa can retain their fertilizing ability after being subjected to widely different preservation methods. The in vitro viability of spermatozoa processed by these methods has not been previously evaluated in detail. Three ejaculates were collected from each of 5 fertile dogs. Each ejaculate was divided into 2 parts and frozen in medium straws according to the 2 methods. Two straws were thawed and examined from each freezing batch. Sperm motility was assessed in the undiluted semen, and in frozen-thawed semen immediately after thawing, and after storage for 3, 6 and 24 h at room temperature (Straw 1) or 1, 2 and 3 h at 37 degrees C (Straw 2, thermoresistance test). The integrity of the sperm plasma membrane was evaluated in undiluted, in equilibrated (diluted and chilled), and in frozen-thawed spermatozoa using fluorophore probes. The acrosome morphology of frozen-thawed spermatozoa was assessed using a commercial stain (Spermac). Motility immediately after thawing was significantly higher with the CLONE method (75.3% [SD = 4.0] for Straw 1 and 73.7% [SD = 3.2] for Straw 2) than with the Andersen method (70.0% [SD = 5.1] and 69.7% [SD = 3.2]). Motility decreased during storage after thawing. Spermatozoa frozen-thawed using the CLONE method showed a significantly lower thermoresistance. The proportion of spermatozoa with intact plasma membrane was not affected by the equilibration procedure used with either method but was significantly decreased (P < 0.001) after thawing with both methods. The percentage of spermatozoa exhibiting changes thought to represent different stages of acrosomal degradation, was 45.7% (SD = 5.3) using the Andersen method and 44.1% (SD = 9,4) using the CLONE method. Both cryopreservation methods thus resulted in high initial post-thaw sperm motility and membrane integrity but low thermoresistance, and under both methods a large proportion of sperm cells were undergoing acrosomal degradation. The methods differed significantly in terms of their effect on sperm motility but not on plasma membrane integrity or acrosomal morphology.     

Is there a relationship between the chromatin status and DNA fragmentation of boar spermatozoa following freezing-thawing?

In this study a radioisotope method, which is based on the quantitative measurements of tritiated-labeled actinomycin D ((3)H-AMD) incorporation into the sperm nuclei ((3)H-AMD incorporation assay), was used to assess the chromatin status of frozen-thawed boar spermatozoa. This study also tested the hypothesis that frozen-thawed spermatozoa with altered chromatin were susceptible to DNA fragmentation measured with the neutral comet assay (NCA). Boar semen was diluted in lactose-hen egg yolk-glycerol extender (L-HEY) or lactose ostrich egg yolk lipoprotein fractions-glycerol extender (L-LPFo), packaged into aluminum tubes or plastic straws and frozen in a controlled programmable freezer. In Experiment 1, the chromatin status and DNA fragmentation were measured in fresh and frozen-thawed spermatozoa from the same ejaculates. There was a significant increase in sperm chromatin destabilization and DNA fragmentation in frozen-thawed semen as compared with fresh semen. The proportions of spermatozoa labeled with (3)H-AMD were concurrent with elevated levels of sperm DNA fragmentation in K-3 extender, without cryoprotective substances, compared with L-HEY or L-LPFo extender. Regression analysis revealed that the results of the (3)H-AMD incorporation assay and NCA for frozen-thawed spermatozoa were correlated. Boars differed significantly in terms of post-thaw sperm DNA damage. In Experiment 2, the susceptibility of sperm chromatin to decondensation was assessed using a low concentration of heparin. Treatment of frozen-thawed spermatozoa with heparin revealed enhanced (3)H-AMD binding, suggesting nuclear chromatin decondensation. The deterioration in post-thaw sperm viability, such as motility, mitochondrial function and plasma membrane integrity, was concurrent with increased chromatin instability and DNA fragmentation. This is the first report to show that freezing-thawing procedure facilitated destabilization in the chromatin structure of boar spermatozoa, resulting in an unstable DNA that was highly susceptible to fragmentation.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

Dietary omega-3 fatty acids (fish oils) have limited effects on boar semen stored at 17 °C or cryopreserved

To evaluate the influence of dietary supplementation of omega-3 polyunsaturated fatty acids (n-3 PUFA) on storage of boar semen, three experiments were conducted: two involved long-term, fresh semen storage (Exp. 1 and Exp. 2), whereas the other involved cryopreservation (Exp. 3). Boars were allocated randomly to three dietary treatments (for 6-7 mo). In addition to a daily allowance of 2.5 kg of a basal diet, they received: 1) 62 g of hydrogenated animal fat (AF); 2) 60 g of menhaden oil (MO), containing 18% docosahexanoic acid (DHA) and 15% eicosapentanoic acid (EPA); or 3) 60 g of tuna oil (TO), containing 33% DHA and 6.5% EPA. In Experiment 1 (n = 26) and Experiment 2 (n = 18), semen was cooled and stored in vitro for several days at 17 °C before assessment, whereas in Experiment 3 (n = 18), viability, motility, acrosomal integrity, susceptibility to peroxidation (LPO), and DNA fragmentation were determined in fresh and frozen-thawed sperm. In Experiment 1, sperm from boars fed TO had better resistance to fresh storage; even after 7 or 9 d of storage at 17 °C, there were more (P = 0.03) motile sperm in boars fed TO (>60%) than in those fed AF or MO. In Experiment 2, fish oil supplementation did not influence any aspect of sperm quality during semen storage (P > 0.10). In Experiment 3, cryopreservation decreased the proportion of motile and viable frozen-thawed sperm as well as acrosomal integrity and increased DNA fragmentation and LPO (P < 0.01) relative to fresh semen, although sperm quality was unaffected by treatments (P > 0.09). In conclusion, although adding fish oil to the diet failed to significantly improve the quality of cryopreserved boar sperm, inconsistent responses of long-term storage of cooled sperm to dietary n-3 PUFA supplementation warrant further investigation.     

Cryogenic preservation of semen from the Aleutian Canada goose (Branta canadensis leucopareia)

Aleutian Canada geese (Branta canadensis leucopareia) were inseminated with frozen-thawed semen containing 6% or 7% dimethylsulfoxide (DMSO) resulting in 32 fertile eggs and 17 goslings; with 7% DMSO, 19 of 31 eggs were fertile. Beltsville Poultry Semen Extender (BPSE), adjusted to 270 ± 30 mOs and 7.5 ± 0.4 pH, was used to dilute semen samples and the DMSO before cryopreservation. About half of the live spermatozoa in the fresh semen (92.9 ± 2.5% live cells, laboratory studies; 87.3 ± 7.3%, insemination trials) survived the freeze-thaw process (46.7 ± 7.8%, laboratory; 33.3 ± 17.8%, insemination trials). Samples of frozen-thawed semen contained a greater percentage of bent spermatozoa (27.1 ± 8.4% of live cells) than fresh semen (14.4 ± 3.0% of live cells). Fecal- and urate-contaminated semen (a common problem when collecting goose semen) reduced the sperm motility score from 3.2 ± 0.6 to 2.7 ± 0.7 and number of live spermatozoa in frozen-thawed semen from 49 ± 9% to 24 ± 18%. Other variables examined that had less of an effect on semen quality included semen extenders, semen holding temperature, dilution and equilibration, relationship between hour of semen collection and level of semen contamination, and the relationship between season and sperm concentration.     

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris–egg yolk extender, equilibrated (4 °C for 4 h) and frozen at −196 °C in LN₂. The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P < 0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P < 0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P < 0.01) reduced after freezing–thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r = 0.81) and high membrane fluidity (r = 0.65), and negatively correlated with cholesterol level (r = −0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.     

In vitro fertilizing capacity of frozen-thawed boar semen

We describe a porcine semen cryopreservation technique and assess the in vitro fertilizing capacity of the frozen-thawed spermatozoa. The thawed spermatozoa did not lose the physiological properties of motility, viability, and acrosome reaction or capacity to fertilize in vitro. Immediately after thawing, the spermatozoa showed 51% mean motility, 60% viability, and 5% induced acrosome reaction. After 2.5 h of incubation in TALP medium, the spermatozoa exhibited 61% motility, 63% viability and 40% induced acrosome reaction. The average in vitro fertilization capacity of thawed spermatozoa was 68% compared with that of spermatozoa from fresh semen (85%). The percentage of polyspermy was highly variable, with frozen-thawed samples ranging from 0 to 28% and fresh samples from 0 to 30%. The results obtained with frozen semen from 5 boars of different breeds did not show considerable variation. This suggests that the freezing-thawing technique is reproducible and adequate for in vitro fertilization.     

Effects of cryo-injury on progesterone receptor(s) of canine spermatozoa and its response to progesterone

The integrity of sperm progesterone (P4) receptor(s) and its response to steroid stimulation might be crucial for the maintenance of sperm fertilizing ability after cryopreservation. The aim of the current investigation was to study the effect of cryo-procedures on canine sperm P4 receptor(s). In addition, alteration of P4 receptor(s) at the molecular level and their functional integrity following cryo-procedures was evaluated. Fresh and frozen-thawed semen samples (n=6 same dogs) after capacitation were treated with 10 microg/mL P4 to induce the acrosome reaction (AR, FITC-PNA staining). Parallel samples were treated with 50% canine seminal plasma (SP) prior to AR induction with P4. The percentages of AR in capacitated fresh and frozen-thawed semen samples after treatment with P4 were 31.0+/-6.7 and 21.6+/-4.1% (P<0.05), respectively. The percentage of AR in fresh and frozen-thawed semen samples pretreated with SP and incubated with P4 was; 11.5+/-4.8 and 16.5+/-2.0% (P<0.05), respectively. The incidence of the spontaneous AR (P>0.05) in fresh and frozen-thawed semen samples at the onset (5.5+/-2.2 and 6.1+/-1.8%; respectively) and after a 2h (9.6+/-5.1 and 10.4+/-2.7%; respectively) capacitation, avoiding P4 stimulation, were not different. The percentage of progesterone-BSA-FITC staining over the acrosomal region was 18.3+/-10.3% in fresh semen, 36.0+/-11.9% in capacitated (P<0.05) and less than 5% in SP treated spermatozoa. This staining was barely visible in frozen-thawed spermatozoa regardless of capacitation status. In western blot analysis, mAb C262 recognized two bands (54 and 65 kDa). Digitonin treated fresh and frozen-thawed spermatozoa, labeled with [3H]-progesterone, revealed that the P4 binding capacity decreased from 6.0+/-4.4 in fresh to 3.0+/-2.1 nM in frozen-thawed spermatozoa. In nearly all samples tested (except one) 65 kDa protein band decreased significantly after freeze-thaw procedures while the 54kDa protein was increased. These results indicate that the reduced incidence of AR in response to P4 in frozen spermatozoa is possibly due to the conformational changes of P4 receptor(s) and/or reduced P4 receptor density derived from freezing injury.     

The uptake of glycerol, alpha-aminoisobutyric acid and 2-deoxy-D-glucose by spermatozoa of a line of chickens selected for fertility of frozen-thawed semen and a control line

The uptake of glycerol, alpha-aminoisobutyric acid and 2-deoxy-D-glucose by fowl spermatozoa was each measured in two trials using seventh generation males of a line selected for duration of fertility of frozen-thawed semen and those of a randomly selected control line. The selected line had significantly (P< 0.01) higher fertility of frozen-thawed and fresh semen than the control line. The association between glycerol level in spermatozoa before freezing and the fertility of frozen-thawed semen was examined. Significantly greater amounts (CPM/10(9) cells) of glycerol, alpha-aminoisobutyric acid and 2-deoxy-D-glucose were taken up by the spermatozoa of the selected line than those of the control line at 15, 30 and 60 min of incubation. Rank correlations between the fertility of frozen-thawed semen and glycerol levels were generally positive but low in magnitudes.     

In vitro fertilization of porcine oocytes with fresh and frozen-thawed ejaculated or frozen-thawed epididymal semen obtained from identical boars

The objective of this study was to compare the in vitro fertilizing capacity of porcine spermatozoa from fresh and frozen-thawed semen and frozen-thawed epididymal spermatozoa obtained from identical boars. Prior to IVF, fresh spermatozoa were capacitated in TCM 199. Frozen semen samples were stored in 0.25-ml plastic straws using a lactose/glycerol/orvus-es-paste extender. Cumulus-oocyte-complexes (COC) obtained from superovulated prepuberal gilts were fertilized in vitro within 2 h after aspiration with one of the semen samples. After final dilution for IVF, frozen-thawed epididymal semen samples showed motility rates (72.2 +/- 5.6%) similar to those of spermatozoa in fresh semen (76.4 +/- 4.5%), while sperm motility decreased in frozen-thawed ejaculated semen (40.2 +/- 9.4%). Considerable individual differences in sperm motility between boars were observed for ejaculated semen but not for epididymal semen. Enhanced fertilizing capacity of frozen-thawed epididymal spermatozoa was confirmed by pronucleus formation and cleavage rates, with significantly more embryos developing to the 2- and 4-cell stages compared with the groups fertilized with fresh or with frozen-thawed ejaculated semen (59.7 vs 14.6 and 16%). In conclusion, consistent in vitro fertilization rates with minimal semen variability are obtained using frozen-thawed epididymal semen. Following a modified freezing protocol, epididymal spermatozoa can easily be frozen in small containers for IVF, with higher resultant motility and fertilization rates than with ejaculated semen.     

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Holstein bulls: effects of cryopreservation and between-bull variation

The aims of the present study were: (1) to determine the existence of sperm subpopulations with specific motility characteristics in fresh ejaculates from Holstein bulls, (2) to investigate the effects of semen cryopreservation and post-thaw incubation on the distribution of spermatozoa within the different subpopulations, and (3) to evaluate the existence of between-bull variation in the sperm subpopulations structure of fresh and frozen-thawed semen. Six ejaculates were collected from each of 9 Holstein bulls and cryopreserved following a standard protocol. Overall sperm motility and the individual kinematic parameters of motile spermatozoa, determined using a CASA system, were evaluated before freezing and after 0, 2 and 4h of post-thaw incubation at 37 degrees C. Data from 16,740 motile spermatozoa, defined by VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF, were analysed using a multivariate clustering procedure to identify and quantify specific subpopulations within the semen samples. The statistical analysis clustered all the motile spermatozoa into four separate subpopulations with defined patters of movement: Subpopulation (Subp. 1) moderately slow but progressive spermatozoa (23.2%), (Subp. 2) highly active but non-progressive spermatozoa (16.0%), (Subp. 3) poorly motile non-progressive sperm (35.5%), and (Subp. 4) highly active and progressive sperm (25.3%). Subpopulations 2 and 4 significantly (P<0.01) decreased during cryopreservation and post-thaw incubation (Subp. 2: 21.1%, 18.1%, 8.7% and 5.9%; and Subp. 4: 34.1%, 20.6%, 15.2% and 7.3%, respectively, for fresh, 0, 2 and 4h post-thaw) whereas Subp. 3 significantly (P<0.01) increased (10.7%, 27.2%, 27.2% and 30.7%, respectively, for fresh, 0, 2 and 4h post-thaw). The frequency distribution of spermatozoa within subpopulations was quite similar for the 9 bulls, either in fresh or frozen-thawed semen, and differences among bulls were mainly due to differences in the Subp. 4. Significant correlations (P<0.01) were found between the proportions of spermatozoa assigned to Subp. 4 in the fresh ejaculates and those in frozen-thawed semen after 0 (r=0.473), 2 (r=0.513) and 4h post-thaw (r=0.450). This indicated that the ejaculates with the highest subpopulations of rapid and progressive sperm were also the most resistant to cryopreservation and showed the best post-thaw sperm longevity.     

Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics

Boar semen is typically collected, diluted and cooled for AI use over numerous days, or frozen immediately after shipping to capable laboratories. The storage temperature and pH of the diluted, cooled boar semen could influence the fertility of boar sperm. Therefore, the purpose of this study was to determine the effects of pH and storage temperature on fresh and frozen-thawed boar sperm motility end points. Semen samples (n = 199) were collected, diluted, cooled and shipped overnight to the National Animal Germplasm Program laboratory for freezing and analysis from four boar stud facilities. The temperature, pH and motility characteristics, determined using computer automated semen analysis, were measured at arrival. Samples were then cryopreserved and post-thaw motility determined. The commercial stud was a significant source of variation for mean semen temperature and pH, as well as total and progressive motility, and numerous other sperm motility characteristics. Based on multiple regression analysis, pH was not a significant source of variation for fresh or frozen-thawed boar sperm motility end points. However, significant models were derived which demonstrated that storage temperature, boar, and the commercial stud influenced sperm motility end points and the potential success for surviving cryopreservation. We inferred that maintaining cooled boar semen at approximately 16 °C during storage will result in higher fresh and frozen-thawed boar sperm quality, which should result in greater fertility.     

Semen cryopreservation of yellow croaker <Emphasis Type="Italic">Larimichthys polyactis</Emphasis>

The effects of various extenders and cryoprotectants on movable spermatozoa ratio (MSR), spermatozoa velocity (SV) and duration of spermatozoa motility (DSM) of post-thawed spermatozoa were examined. The MSR, SV and DSM of post-thawed sperm in artificial seminal plasma (ASP) extender were higher than those in marine fish Ringer’s solution (MFRS) extender (P < 0.01) and was not significantly different from that of fresh sperm. No significant differences were observed in the motility parameters between fresh spermatozoa and frozen-thawed spermatozoa cryopreserved with ASP extender supplement 10% EG (ethylene glycol) cryoprotectant. Using the above method, yellow croaker semen was cryopreserved with extender ASP and 10% EG. As a result, at the spermatozoa/egg ratio of 100,000:1, the fertilization rate and hatching rate of the frozen-thawed spermatozoa cryopreserved for 1 week or 1 year in liquid nitrogen (45.7 ± 3.2% and 27.2 ± 5.0% or 37.5 ± 4.4% and 27.2 ± 5.0%) were similar to that of fresh spermatozoa (51.0 ± 3.1% and 36.7 ± 2.2%). There was a small alternation of shape in cryopreserved spermatozoa compared with fresh spermatozoa. In conclusion, the optimal conditions for yellow croaker semen cryopreservation are ASP extender supplement 10% EG cryoprotectant. This is the first report on semen cryopreservation of yellow croaker Larimichthys polyactis.