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.     

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.     

Post-thaw viability of bull AI-doses with low-sperm numbers

Use of AI-doses containing low-sperm numbers are increasingly been used to optimise use of elite bulls as well as to accommodate an eventual wider application of sex-sorted semen. Since spermatozoa might, however, suffer from high extension rates, thus compromising fertility, this study evaluated the post-thaw sperm quality of semen from commercial progeny-tested, high-ranked AI-sires whose semen was within acceptable limits of normality, frozen in a split-design to 15 (control, 15M) or 2 x 10(6) total spermatozoa (treatment, 2M) per straw. Assessment post-thaw included computer-evaluated sperm motility (CASA), membrane integrity (SYBR-14/PI), membrane stability (Annexin-V/PI), acrosome integrity (Carboxy-SNARF-1/PI/FITC-PSA), and chromatin integrity (AO of in situ acid-induced DNA denaturation). High extension did not affect the proportions of linearly motile spermatozoa, of membrane integrity or stability nor chromatin integrity, immediately post-thaw. However, high extension clearly affected linear sperm motility following incubation at 38 degrees C for 30 min, sperm viability when assessed by SNARF and, particularly, acrosome integrity of the otherwise viable spermatozoa. Individual sire variation was evident. Fertility was preliminarily evaluated for one of the less affected bulls in a blind field trial. A total of 109 dairy cows were randomly inseminated with 15M or 2M-straws without differences in pregnancy rate between them (47% versus 43%). This similarity in fertility rates, confirmed the in vitro methods used were appropriate for identifying cryosurvival and further suggested the site of sperm deposition was not crucial for the fertility of low-sperm AI-numbers for this particular sire. However, the inter-bull variation seen calls for caution when cryopreserving low concentrations of bull spermatozoa with conventional freezing protocols.     

Supplementation of the thawing medium with reduced glutathione improves function of frozen-thawed goat spermatozoa

Sperm cryopreservation represents a useful tool in the management of reproduction in goat production. However, freezing and thawing produce physical and chemical stress on the sperm membrane that reduces their viability and fertilizing ability. In this study, firstly we evaluated the effects of reduced glutathione (GSH, 1 and 5 mM) supplementation of the thawing extender on parameters of frozen-thawed goat spermatozoa. We used a set of functional sperm tests that included sperm motility assayed by computer-assisted semen analysis (CASA), membrane lipid packing disorder, spontaneous acrosome reaction, free radical production (ROS generation) and sperm chromatin condensation. The main findings from this study were that addition of GSH to the thawing medium resulted in: (1) a higher motility and progressive motility; (2) a higher number of non-capacitated viable spermatozoa; (3) higher number of viable spermatozoa with intact acrosome; (4) a reduction in ROS generation and (5) lower chromatin condensation. In a second study, the additions of reduced (GSH, 5 mM) or oxidized glutathione (GSSG, 2.5 mM) to the thawing media were evaluated. We confirmed the protective effect of GSH on the sperm functionality. The addition of GSSG to the thawing media was less protective to sperm functions compared to GSH. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen goat spermatozoa. The information derived from this study suggests the importance of oxidative stress as responsible for cryo-injury to spermatozoa and opens new windows to explore the practical application of antioxidants to improve the quality of post-thaw goat semen.     

Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozen-thawed stallion semen

The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E). Sperm motility, sperm chromatin structure, membrane integrity and mitochondrial membrane potential were studied in filtered and non-filtered spermatozoa. Single-layer centrifugation (SLC) using Androcoll-E significantly improved all the sperm parameters studied, implying SLC may be a simple approach to improve the quality of frozen-thawed (FT) spermatozoa for AI.     

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008–2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24 h at 17 °C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P < 0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season’s influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.     

Membrane status of boar spermatozoa after cooling or cryopreservation

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.     

Mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality

Human spermatozoa cryopreservation is an important means of assisted reproductive technology and male fertility preservation. Although this technique is particularly useful, sperm cryopreservation significantly reduces the quality of spermatozoa after freezing and thawing. The objective of the study is to examine the efficacy of mitochondria-targeted antioxidant MitoTEMPO in improving sperm quality during semen cryopreservation processes. Semen samples were collected and cryopreserved in extenders containing different concentrations (0.0, 0.5, 5, 50, and 500 μM) of MitoTEMPO. Sperm motility, viability, membrane integrity, mitochondrial membrane potential and antioxidant activities were measured and analyzed. The results showed that the addition of MitoTEMPO (5–50 μM) significantly improved post-thaw sperm motility, viability, membrane integrity and mitochondrial membrane potential (P < .05). Meanwhile, antioxidant enzymes activities were enhanced and MDA content were decreased in the group supplemented with MitoTEMPO. In conclusion, mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality and antioxidant enzymes profile.     

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing–thawing caused a significant decrease (P < 0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P < 0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P < 0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.     

Influence of thawing method on motility, plasma membrane integrity and morphology of frozen-thawed stallion spermatozoa

Different thawing methods are used for stallion semen, however, it is unclear which method is the optimal one. To determine if the thawing temperature has an effect on semen quality, we compared 2 thawing temperatures, 75 degrees C and 37 degrees C. The following parameters were used to measure sperm quality: sperm motility, sperm viability, plasma membrane integrity and sperm morphology. Twenty-three ejaculates from 10 Dutch Warmblood stallions were thawed either at 37 degrees C for 30 sec or at 75 degrees C for 7 sec. Sperm motility was evaluated by a Hamilton Thorn Motility Analyser. Plasma membrane integrity and sperm viability were evaluated by using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-1 probe. The eosinaniline blue staining method was used to evaluate the percentage of live and dead cells, as well as sperm morphology. There was no significant difference (P = 0.84) between sperm motility after thawing at 37 degrees C and 75 degrees C. There was also no significant difference (P = 0.053) between the percentage of live spermatozoa using the calcein AM/ethidium homodimer stain after thawing at 37 degrees C and 75 degrees C. There was, however, a significant difference (P = 0.032) between the percentage of live spermatozoa using the eosin-aniline blue stain after thawing at 37 degrees C compared with that at 75 degrees C. In conclusion, our laboratory results indicated that stud farms using frozen semen should thaw the straws at 37 degrees C instead of 75 degrees C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and has to be timed very carefully to avoid damage to the spermatozoa.     

Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time

Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 10⁶ spermatozoa/mL in a TES–Tris–Fructose–based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at −196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin–fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.     

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10⁸ cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10⁸ cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze–thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.     

Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P < 0.05). Fructose was inferior for protecting sperm during cryopreservation when compared to sorbitol and glucose (P < 0.05). Although the viability, motility and acrosome integrity of sperm cryopreserved with a glucose-containing extender did not significantly differ from sperm frozen in the sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender.     

Deep freezing of concentrated boar semen for intra-uterine insemination: effects on sperm viability

The use of deep-frozen boar semen for artificial insemination (AI) is constrained by the need for high sperm numbers per dose, yielding few doses per ejaculate. With the advancement of new, intra-uterine insemination strategies, there is an opportunity for freezing small volumes containing high sperm numbers, provided the spermatozoa properly sustain cryopreservation. The present study aimed to concentrate (2 x 10(9) spz/mL) and freeze boar spermatozoa packed in a 0.5 mL volume plastic medium straw (MS) or a multiple FlatPack (MFP) (four 0.7 mL volume segments of a single FlatPack [SFP]) intended as AI doses for intra-uterine AI. A single freezing protocol was used, with a conventional FlatPack (SFP, 5 x 10(9) spz/5 mL volume) as control. Sperm viability post-thaw was monitored as sperm motility (measured by computer-assisted sperm analysis, CASA), as plasma membrane integrity (PMI, assessed either by SYBR-14/PI, combined with flow cytometry, or a rapid hypo-osmotic swelling test [sHOST]). Sperm motility did not differ statistically (NS) between test-packages and control, neither in terms of overall sperm motility (range of means: 37-46%) nor sperm velocity. The percentages of linearly motile spermatozoa were, however, significantly higher in controls (SFP) than in the test packages. Spermatozoa frozen in the SFP (control) and MFP depicted the highest PMI (54 and 49%, respectively) compared to MS (38%, P < 0.05) when assessed with flow cytometry. In absolute numbers, more viable spermatozoa post-thaw were present in the MFP dose than in the MS (P < 0.05). Inter-boar variation was present, albeit only significant for MS (sperm motility) and SFP (PMI). In conclusion, the results indicate that boar spermatozoa can be successfully frozen when concentrated in a small volume.     

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.     

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.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

Effect of lactose and glycerol on the motility, normal apical ridge, chromatin condensation and chromatin stability of frozen boar spermatozoa

The effect of lactose and glycerol concentration, as well as the equilibration time with glycerol was studied on motility, normal apical ridge (NAR), and chromatin state of boar spermatozoa after the freezing and thawing process. In the first experiment, samples were frozen in first and second extenders containing different concentrations of lactose (11, 12 and 14%). In the second experiment samples were frozen using second extenders with different concentrations of glycerol (4, 6, 8 and 10%) and were incubated at 5 degrees C for 0 and 30 min. Motility, motility after caffeine treatment, NAR, chromatin condensation and stability (susceptibility to de-condense after heparin treatment) were evaluated. The results indicated that freezing spermatozoa in extenders with increasing concentrations of lactose adversely affected motility but provided a protective effect on acrosomes. Increased lactose concentration induced higher chromatin condensation but maintained the same stability. Increasing the glycerol concentration in the second extender from 4-6 to 8% led to higher motility and NAR as well as lower chromatin condensation and stability. When 30 min equilibration time was allowed after dilution with the same extenders, spermatozoa showed higher NAR and lower chromatin condensation and stability. The longer equilibration time was detrimental for motility when freezing in the 8% glycerol extender but favourable when using the 4% glycerol extender. Compared to the 8% glycerol, spermatozoa frozen in the 10% glycerol extender showed similar motility and increased chromatin condensation and stability, as well as low values of NAR that did not improve by longer incubation time.     

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.     

Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation

We investigated the effect of supplementing extended boar semen with different amounts of hyaluronan (HA) prior to freezing on post-thaw sperm characteristics. Using a split sample design, the effect of HA at a final concentration of 500 or 1000 microg/ml semen on post-thaw motility parameters, and membrane lipid architecture status assessed by merocyanine-540/YOPRO-1 and flow cytometry were evaluated. HA-supplementation improved motility parameters (P < 0.05 to P < 0.001) and decreased the percentage of hyperactivated spermatozoa (P < 0.05). HA-supplemented samples had more spermatozoa showing high lipid membrane stability as assessed with merocyanine-540. In conclusion, HA appeared to preserve post-thaw spermatozoa viability in vitro and maintained membrane stability after cryopreservation.