Cryopreserved Bull Spermatozoa Research Articles

The advantages of low-density lipoproteins in the cryopreservation of bull semen

Egg low-density lipoprotein (LDL) was added at concentrations (w/v) of 7%, 8% or 9% to the extenders used to freeze bull semen and its effects on seminal parameters and anti-oxidant activities of frozen–thawed sperm were assessed. Analysis of data showed that sperm exposed to 8% LDL exhibited the greatest percentages of sperm motility, acrosome integrity and membrane integrity, compared to the control which differed from the treatment groups by replacing LDL with 20% egg yolk ( P < 0.05). No difference was observed for membrane integrity between 8% and 9% LDL groups ( P > 0.05). The extender supplemented with LDL did not exhibit improvement in SOD levels. However, 8% LDL group favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison to other groups (7%, 9% LDL and the control) ( P < 0.05). No difference was observed for CAT activity between 9% LDL and the control group. In conclusion, sperm cryopreserved in the extender containing 8% LDL in place of egg yolk exhibited the greatest percentages of post-thaw sperm motility, acrosome integrity and membrane integrity, in comparison with the control, and favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison with other groups. The replacement of egg yolk by LDL in the composition of extenders was beneficial for bull sperm cryopreservation.

Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30–60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 × 10 6 sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 × 10 6 sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 × 10 6 sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.

Effects of different dilution levels on bull- and ejaculate related variability of plasmamembran integrity, acrosomal damage and DNA-integrity of cryopreserved bull spermatozoa

In this study the effects of three different dilution levels (60, 30 and 15 Mio/ml) on quality of cryopreserved bovine sperm were investigated. Particularly it was tested, if effects of dilution in regard to sperm quality were related to the factors bull or ejaculate. The following parameters were analysed in four ejaculates each of 42 bulls: percentage of plasma membrane intact sperm (PMI), percentage of sperm with acrosomal damage (AS) as well as percentage of sperm with a high DNA-fragmentation index (DFI%) following cryopreservation. PMI-values decreased (p < 0.05) with increasing degree of dilution. AS-values were not influenced (p > 0.05) by dilution level. There was a decline in DFI% (p <0.05) with increasing dilution of the ejaculate. Coefficient of variation for PMI, AS and DFI% were not (p > 0.05) influenced by dilution. The effects of the factor ejaculate on the variabilities of PMI and AS were higher than the effects of the factor bull. Variability of DFI% depended at the highest dilution level more on the factor bull and in the lowest dilution level more on the factor ejaculate. In conclusion, these results show that dilution of sperm has positive as well as negative effects on sperm quality. With increasing dilution the effect of the factor bull on plasma membrane- and acrosomal integrity.

Peroxynitrite participates in mechanisms involved in capacitation of cryopreserved cattle

The effect of peroxynitrite (ONOO −) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from five bulls was incubated in Tyrode's albumin lactate pyruvate (TALP) medium in the presence of heparin (10 IU/ml), sodium nitroprusside (SNP, 50 nM), a nitric oxide donor or 3-morpholinosydnonimine (SIN-1, 1–20 μM), a ONOO − donor. The participation of ONOO − was evaluated at 15, 30 and 45 min and confirmed by using a specific scavenger, uric acid (2–20 mM). Spermatozoa capacitated with SIN-1 were incubated with ovarian follicular fluid of cattle to evaluate their ability to undergo acrosome reaction. The role of ONOO − during capacitation induced by heparin or nitric oxide was evaluated by the addition of uric acid. The participation of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK) in capacitation induced by ONOO − was evaluated by incubation with specific inhibitors (50 μM H-89, 0.1 μM bisindolylmaleimide I, and 3 μM genistein, respectively). Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC) and true acrosome reaction was determined by trypan blue and Differential-Interferential Contrast (DIC). SIN-1 concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of 10 μM SIN-1 treatment (23 ± 2%) were significantly greater with respect to the control (4.6 ± 1.62%). At 15 min of incubation the greatest capacitation was observed ( P < 0.05), reaching a plateau between 15 and 45 min. Follicular fluid induced acrosome reaction in spermatozoa previously capacitated with 10 μM SIN-1 ( P < 0.05). Uric acid prevented SIN-1-induced capacitation and significantly diminished capacitation induced by heparin or SNP. The addition of PKA and PKC inhibitors failed to modify the capacitation induced by SIN-1 (27.4 ± 3.85 and 24.8 ± 4.75, respectively). Genistein, a PTK inhibitor, produced a significant capacitation decrease (8.6 ± 5.5%). These results indicate that endogenous ONOO − may be generated during heparin- or SNP-induced capacitation. Exogenous ONOO − acts as a capacitation inducer and involves the participation of PTK, as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.

259 ADDITION OF CHOLESTEROL TO FLOW-SORTED BULL SPERMATOZOA TO ENHANCE CRYOSURVIVAL DOES NOT AFFECT THE ACROSOME REACTION

Treating bull sperm with cholesterol-loaded cyclodextrins (CLCs) improves post-thaw motility and viability (Purdy and Graham 2004 Biol. Reprod. 71, 522–527). Unpublished research in our laboratory demonstrated similar improvements for cryosurvival of CLC-treated sex-sorted bull sperm. Our objective was to determine the effects of CLCs on the dilauroylphosphatidylcholine (PC12)-induced acrosome reaction of flow-sorted and non-sorted (control), frozen/thawed bull sperm. Sperm concentrations (3 ejaculates, 4 bulls) were adjusted to 200 × 106 sperm mL–1 with TALP without Ca++. Sperm for sorting were stained (63 μm Hoechst 33342, 34.5°C, 45 min), and then incubated an additional 15 min with 2.5 mg CLC in TALP (+CLC) or an equivalent volume of TALP without CLCs (–CLC), adjusted to 100 × 106 sperm mL–1 with 4% egg yolk TALP containing 0.002% food dye, filtered, and ‘bulk’ sorted (Schenk et al. 1999 Theriogenology 52, 1375–1391) into 2.0 mL aliquots of 20% egg yolk Tris buffer. Controls were extended in 20% egg yolk Tris A to result in a final concentration of 21 × 106 sperm mL–1. All samples were cooled to 5°C over 90 min, and Tris buffer with (controls) or without (flow-sorted) egg yolk (12% glycerol) was added (v/v). Sorted samples were centrifuged (850g, 20 min), and pellets were suspended in 20% egg yolk/6% glycerol Tris, resulting in 21 × 106 sperm mL–1. Sperm were loaded into 0.25-mL straws and frozen over LN2 vapor. Straws were subsequently thawed (30 s, 37°C), and 25 million sperm/treatment were centrifuged through 60% Percoll® to remove egg yolk. Pellets were adjusted to 20 × 106 sperm mL–1 with TALP containing 2.0 mm Ca++. Aliquots (30 μL) were stained with propidium iodide (PI) and fluorescein isothiocyanate–peanut agglutinin (FITC-PNA); treated with 0, 25, or 35 μL PC12; and brought to a constant volume (300 μL) with TALP. TALP (30 μL) containing 42 mm Ca++ was added, resulting in final concentrations of 0, 68, and 95 μm PC12 and 6 mm Ca++. Following incubation (38°C, 35–40 min) 50 000 sperm were analyzed on an SX MoFlo® (Dako, Fort Collins, CO, USA) to determine the percentage of live acrosome-reacted sperm/live sperm(PI –ve, FITC +ve). There were no differences between CLC-treated and non-treated sorted or non-sorted sperm (Table 1) suggesting that, as previously shown for non-sorted sperm (Purdy and Graham 2004), treatment of flow-sorted sperm with CLCs does not interfere with the progression of the acrosome reaction. Table 1. Effect of treatment with CLCs on % live acrosome-reacted (mean ± SEM) non-sorted and flow-sorted cryopreserved bull spermatozoa

Comet assay of fresh and cryopreserved bull spermatozoa

In this study, we describe DNA fragmentation of fresh and cryopreserved bull spermatozoa using the comet assay. Cryopreservation caused a significant but low (3.8%) decrease in the percentage of DNA in the comet head and an increase (5.3%) in the tail length. Our results suggest that in addition to motility and viability, low levels of DNA fragmentation after cryopreservation is a characteristic of bull spermatozoa and can be a part of remarkable cryoresistance of bull spermatozoa.

A comparison of the protective action of added egg yolks from five avian species to the cryopreservation of bull sperm

Cryopreservation of domestic animal sperm has been widely used for artificial insemination (AI), and egg yolk is one of the most commonly used cryoprotectants during the freezing–thawing process. The objectives of this study were to compare the effectiveness of egg yolk from five avian species (domestic chicken, domestic duck, domestic goose, Japanese quail or domestic pigeon) and to optimize the concentration of egg yolk on the cryopreservation of bull sperm in terms of frozen–thawed sperm progressive motility and viability. The results were two-fold. First, they showed that pigeon egg yolk provided the best cryoprotective effects on the cryopreservation of bull sperm, compared with egg yolk of chicken, quail, goose or duck. Second, the best concentration of pigeon egg yolk in extender was 20% during cryopreservation among five concentrations of 5, 10, 20, 30 or 40%. The results suggest that pigeon egg yolk could be used as an alternative to chicken egg yolk in extender but requires further testing in fertility trials.

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.

124. A novel method for separating motile sperm using cryopreserved bull sperm and a microfluidic device

124. A novel method for separating motile sperm using cryopreserved bull sperm and a microfluidic device

Impact of antifreeze proteins and antifreeze glycoproteins on bovine sperm during freeze-thaw

There are no reports on the use of antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) for the use of bull sperm cryopreservation despite studies in the ram, mouse and chimpanzee. The effect of freezing and thawing on bull sperm viability, osmotic resistance and acrosome integrity were observed following the addition of AFP1, AFPIII and AFGP at four concentrations (0.1, 1, 10 and 100 μg/ml). In a second part of the experiment, fluorescein was conjugated to the AFPs and AFGP and observations were made using fluorescence microscopy to determine whether binding occurred between the sperm cell membranes and the proteins. In the final part of the study the cryopreservation media were cooled in the presence of the AFPs and AFGPs at the four concentrations on a cryomicroscope to mimic similar cooling curves as those used in the presence of sperm. Following freeze-thaw, AFPI resulted in increased osmotic resistant cells at 0.1–10 μg/ml compared to the control ( P < 0.01). AFPI and AFPIII did bind to the sperm cells. There was no visual difference in ice structure between the control, AFPIII and AFGP but AFPI resulted in parallel crystals at 0.1, 1 and 10 μg/ml. We suggest that the increased osmotic resistance in the spermatozoa cryopreserved in AFPI is due to the cells orientating between the ice crystals, reducing mechanical stress to the cell membrane. Previous research has shown that osmotic resistance correlates with bull fertility, suggesting that bull spermatozoa cryopreserved in the presence of AFPI may have increased fertility in vivo.

Effect of sex-sorting on the ability of fresh and cryopreserved bull sperm to undergo an acrosome reaction

Previous studies indicate that sex-sorted sperm exhibit different physiology, including fertilizing capacity, from non-sorted sperm. However, differences between X- and Y-bearing sperm in their ability to undergo an acrosome reaction have never been investigated. This study determined the ability of non-sorted and sex-sorted sperm to undergo the acrosome reaction prior to and after cryopreservation. Sperm were treated with dilauroylphosphatidylcholine (PC12) to induce the acrosome reaction and the percentages of live-acrosome-reacted sperm and dead sperm were evaluated. The X- and Y-bearing sperm reacted similarly to the PC12 treatment, regardless of whether sperm were assessed prior to or after cryopreservation. Fresh control sperm exhibited lower percentages of live sperm (60%) than either X- or Y- sorted sperm (69–74%, P<0.05). Percentages of live control sperm were also lower after thawing (29–35%) than sex-sorted sperm (55–58%, P<0.05). Control and sex-sorted fresh sperm responded similarly to PC12 treatment. However, sex-sorted cryopreserved sperm exhibited higher percentages of live-acrosome-reacted sperm (23%) than control sperm (9%, P<0.05) after 40min without PC12 treatment. In addition, cryopreserved control sperm treated with 79μM PC12 exhibited higher percentages of live-acrosome-reacted sperm than sex-sorted sperm. In conclusion, X- and Y-bearing sperm responded similarly to PC12 treatment. In addition, fresh sexed and non-sorted sperm responded similarly to PC12 treatment. However, cryopreserved sex-sorted sperm underwent an acrosome reaction more rapidly in the absence of PC12 (over a 40min period) than the non-sorted sperm. Therefore, sex-sorting induced changes in sperm membranes that accelerated the acrosome reaction process in sperm after cryopreservation.

Nitric oxide-induced capacitation of cryopreserved bull spermatozoa and assessment of participating regulatory pathways

The effect of nitric oxide (NO ) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from four bulls were incubated in TALP medium in the presence of heparin (10 IU/ml) or sodium nitroprusside (SNP, 0.05–100 μM), a NO donor. The participation of NO was confirmed by the use of scavengers, i.e. methylene blue (50 100 μM) and hemoglobin (20–40 μg/ml). The role of nitric oxide synthase in heparin-induced capacitation was evaluated using enzyme inhibitors Nω-nitro- l-arginine methyl ester ( l-NAME) and Nω-nitro- l-arginine ( l-NA) in concentrations ranging from 1 to 500 μM. The effects of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK), on NO -induced capacitation were evaluated by incubation with specific inhibitors of these enzymes (H-89, 50 μM; bisindolylmaleimide I, 0.1 μM and genistein, 3 μM). The role of hydrogen peroxide or superoxide anion in NO -induced capacitation was evaluated by incubation with catalase (20–100 μg/ml) or superoxide dismutase (SOD, 0.05–0.5 mg/ml), respectively. Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC). SNP concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of the 0.05 μM SNP treatment (31 ± 5.15%) were similar to those of heparin treated samples (33 ± 4.27%). Inhibitors of nitric oxide synthase (NOS) diminished capacitation percentages in a dose-dependent manner as did the addition of NO scavengers ( P < 0.05). The presence of PKA, PKC and PTK inhibitors likewise decreased capacitation percentages (6.25 ± 0.71, 12.75 ± 1.41, 9.00 ± 1.41%, respectively). The presence of catalase or SOD in the incubation medium had no effect on capacitation percentages. These results indicate that NO may be generated by a sperm NOS during heparin-induced capacitation and that exogenous NO acts as a capacitation inducer and involves the participation of PKA, PKC and PTK as part of the intracellular mechanisms that lead to capacitation in cryopreserved bull spermatozoa.

Thiols prevent H 2O 2-mediated loss of sperm motility in cryopreserved bull semen

We previously showed that cryopreservation of bull spermatozoa in egg yolk Tris extender (EYTG) significantly reduced the intracellular level of thiols. Other studies showed the beneficial effects of adding antioxidants to cryopreserved bull spermatozoa. These studies led us to investigate the effects of various thiols, an important class of antioxidants, on sperm motility of cryopreserved bull semen in a commonly used extender, EYTG. Sperm motility was analyzed by computer-assisted semen analysis (CASA). After thawing,a diluted pool of bull semen was incubated at 38.5°C in airtight tubes with the following thiols for 6 hours: glutathione (GSH/GSSG), cysteine, N-acetyl-L-cysteine (NAC) and 2-mercaptoethanol in the presence or absence of oxidative stress. The oxidative stress was caused by adding H 2O 2 (100 μM) to diluted semen. Incubation of diluted bull semen in EYTG at 38.5°C over a period of 6 h decreased sperm motility by ∼9 fold from the start (72 ± 3, mean ± SEM, n=4) to the end (9 ± 4, n=4) of the incubation. We found that all thiols to a concentration above 0.5 mM maintained high sperm motility for 6 h in the absence of an external source of oxidative stress (52 ± 4, for 4 thiols). However, one mM of each thiol was required to efficiently protect sperm motility in the presence of 100 μM of H 2O 2 for 6 h. We also found that the GSH concentration in diluted semen was too low (μM) to adequately supply exogenous addition of 72 U/mL of glutathione peroxidase (GPx), an enzyme that detoxifies H 2O 2 and hydroperoxides using GSH as a cofactor. In fact, a better protection of sperm motility could be achieved with only 5 U/mL of GPx and 0.1 mM of GSH added to diluted semen. Our results also demonstrated that added GSSG (0.5 mM) in diluted semen was not regenerated efficiently to GSH over 6 h. The latter result indicated in the extender that the glutathione redox-cycle was deficient. Therefore, deleterious effects on sperm motility after cryopreservation in EYTG can be counteracted by adding various thiols and mM concentration

Lipid composition of virosomes modulates their fusion efficiency with cryopreserved bull sperm cells.

Virosomes derived from different fusogenic enveloped viruses have been generated for potential application in gene targeting to sperm cells. Comparative characterization of reconstitution products revealed that virosomes derived from influenza viruses are superior to those generated from Sendai viruses, with respect to the fusion rates with cryopreserved bull sperm cells and to sperm cell vitality after fusion. Modulation of the lipid composition during virosome reconstitution affects fusion sites on target sperms and allows optimization of the fusion rate and sperm cell vitality. A fluorescence-based microscopic fusion assay combined with a vital cell stain revealed that about 90% of sperm cells fused with influenza virosomes containing exogenous cholesterol, sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. About 85% of the fused sperm cells remained vital. Such optimized influenza-derived virosomes provide the basis for ongoing experiments, which aim at eventually generating biologically active transgenic sperms.

Chlortetracycline staining patterns of frozen-thawed bull spermatozoa treated with beta-cyclodextrins, dibutyryl cAMP and progesterone.

Efforts to achieve complete chemical definition of media used for in vitro capacitation of bovine spermatozoa including removal of heparin purified from porcine intestinal mucosa are presented. Fluorescent staining with chlortetracycline (CTC), known to reflect changes coincident with sperm capacitation in certain species, was studied following treatments of frozen-thawed bull spermatozoa with beta-cyclodextrins, dibutyryl cAMP (dbcAMP) and progesterone in comparison with heparin. The CTC staining patterns (F, B and AR) were confirmed to correlate with known conditions that effectively prepare cryopreserved bull spermatozoa for fertilisation in vitro. In the absence of glucose, the routinely employed heparin-containing capacitating medium caused an increase in spermatozoa displaying the AR pattern. Both progesterone (100 microM) and dbcAMP (0.01-0.1 mM) were able to increase the proportion of B pattern stained sperm cells more than after exposure to control (mDM) conditions without a significant reduction in motility. Exposure to either dbcAMP or beta-cyclodextrins was accompanied by an increase in proportions of spermatozoa displaying the AR pattern over those seen in controls. Exposure to beta-cyclodextrins did not increase the proportion of B pattern stained spermatozoa. Comparison of spermatozoa from two bulls revealed differential responses of spermatozoa from different males to treatments with heparin and progesterone. In vitro fertilisation results demonstrated that previously cryopreserved bull spermatozoa could be capacitated in chemically defined conditions devoid of heparin or other biological components.

Osmotic Effects on Volume and Motility of Bull Sperm Exposed to Membrane Permeable and Nonpermeable Agents

Factorially arranged experiments were designed to study prefreeze packed cell volume (PCV) changes and associated percentages of motile and unstained bull sperm in simple macromolecule-free Tyrode's solution and egg yolk–Tris (EYT), varying in osmolarity, and with addition of rapidly permeating cryoprotectants, glycerol and 1,2-propanediol, and nonpermeating substances, sucrose and NaCl. The percentage of motile and unstained sperm was assessed after resuspending sperm in 300 mOsm/L Tyrode's solution. At 25°C PCV increased in Tyrode's solution as osmolarity was decreased from 250 to 150 mOsm/L and decreased as Tyrode's solution was increased to 400 mosmol/L. The relationship of PCV to the reciprocal of the osmolarity was essentially linear over the range of 150 to 400 mOsm/L, but PCV did not decrease further in solutions ranging from 500 to 1000 mOsm/L. The percentage of motile sperm declined to zero in Tyrode's solution at 700 mOsm/L, but 40% of the sperm were still unstained in 1000 mOsm/L solutions. The addition of glycerol or 1,2-propanediol had little effect on PCV. With glycerol or 1,2-propanediol added to 308 mOsm/L Tyrode's solution to give a total of 1267 mOsm/L, there were 49 and 56% motile sperm, respectively, compared to 1% with NaCl added to give 787 mOsm/L. The PCV and percentage of motile sperm suspended in EYT responded to osmotic changes similar to those reported for Tyrode's solution at both 25 and 5°C. Some sperm remained motile after initial exposure to 800 mOsm/L solutions. These findings may have application in improving bull sperm cryopreservation.

Effects of Trehalose and Sucrose, Osmolality of the Freezing Medium, and Cooling Rate on Viability and Intactness of Bull Sperm after Freezing and Thawing

The influence of the presence of trehalose or sucrose, the osmolality of the freezing medium, and the cooling rate on success of cryopreservation of bull sperm were investigated. Bull semen was frozen at four different cooling rates varying from 40 to 300°C/min in standard Tris–egg yolk medium and in hypertonic or isotonic media containing 0.2Mtrehalose or sucrose. Sperm motility, H33258 exclusion, and acrosome morphology were evaluated. In the hypertonic media the motility was almost completely inhibited, but could partly be restored by returning to isotonic conditions. Medium composition and cooling rate had significant effects, with a strong interaction of medium × cooling rate. The optimal cooling rate was found to be between 76 and 140°C/min. At the highest cooling rate, both the presence of the sugars and the high osmolality of the hypertonic media conferred significant protection against fast-cooling damage, sucrose being significantly better than trehalose. At all other cooling rates the medium differences were less pronounced, but the isotonic sugar media were generally superior to standard medium as to preservation of acrosome intactness and H33258 exclusion. This indicates that the isotonic sugar media could enable some improvement of cryopreservation of bull semen over the currently used Tris–egg yolk medium. Further improvement of sperm survival at the AI station should be possible by implementation of controlled rate freezing, using a cooling rate in the optimal range between 76 and 140°C/min.

Changes in ATP level and iron-induced ultra-weak photon emission in bull spermatozoa, caused by membrane peroxidation during thermal stress.

ATP level, cell motility and viability, oxygen uptake, pyruvate kinase activity, and ultra-weak photon emission (UPE) induced by red-ox Fe(2+)-ascorbate cycling system were studied in fresh, in previously equilibrated in a glycerol diluent, and in cryopreserved bull spermatozoa, exposed to thermal stress by incubation of the cells at 44 degrees C. A sharp drop in motility and viability of fresh spermatozoa and even more so, of equilibrated and cryopreserved cells was accompanied by accumulation of ATP. When cell movement was totally inhibited, ATP utilization was decreased, while chemical energy continued to be produced by cell pyruvate kinase, one of the key glycolytic enzymes, which in spermatozoa is very active (6500 IU/g protein) and insensitive to feed-back inhibition by excess of ATP and L-cysteine. Accumulation of ATP during incubation at 44 degrees C in 0.9% NaCl was accompanied by rapid decrease in oxygen consumption by fresh spermatozoa and an increase in Fe(2+)-ascorbate induced UPE, followed by a sharp decrease in ATP level observed at the end of induced UPE measurement. The increase in photon emission due to lipid peroxidation was highly correlated with the increase in cell ATP level caused by thermal stress.

Ca2+ Regulation by Cryopreserved Bull Spermatozoa in Response to A23187

The regulation of intracellular Ca2+ by fresh and cryopreserved bull spermatozoa from the same ejaculates (n = 5) was investigated using the fluorescent Ca2+ indicator, indo-1. Relative internal Ca2+ levels of the spermatozoa were monitored for 30 min prior to the addition of phosphate-buffered saline (PBS), mM Ca2+ , the Ca2+ ionophore A23187 (0.1 μM), or Ca2+ + A23187; during the additions the levels of intracellular Ca2+ were observed in detail. After these additions, changes in internal Ca2+ levels were monitored for 120 min. The initial intracellular Ca2+ levels in the cryopreserved spermatozoa were greater than those in fresh (1.05 ± 0.03 vs 0.97 ± 0.03 Ca2+ units, P = 0.0001 ). The addition of Ca2+ + A23187 induced elevated cellular Ca2+ in both fresh (P = 0.0177) and cryopreserved spermatozoa (P ⩽ 0.0588) within 5 s. Ca2+ alone did not differ from Ca2+ + A23187 in increasing Ca2+ in cryopreserved spermatozoa (P = 0.2225); fresh spermatozoa were slower to respond to exogenous Ca2+ alone (P = 0.0438). At the start of the post-treatment 120 min, cryopreserved spermatozoa had more internal Ca2+ than the corresponding fresh samples (P ≤ 0.0088) except for those exposed to both Ca2+ + A23187 (P = 0.2918). All spermatozoa increased internal Ca2+ over the post-treatment time except for the cryopreserved PBS controls, and these cryopreserved controls differed from the fresh controls in accumulation of Ca2+ (P = 0.0620). None of the Ca2+/A23187 treatments induced different rates of change in internal Ca2+ over time in fresh cells (P = 0.3142). The late of Ca2+ accumulation by cryopreserved spermatozoa in the presence of exogenous Ca2+ exceeded controls (23.12 ± 6.75 vs 8.11 ± 6.75, P = 0.0005), but A23187, with or without Ca2+, had no effect (P ⩾ 0.097). Following Ca2+ measurements, the viability of the cryopreserved samples was reduced (P ⩽ 0.015) in all but the Ca2+-treated spermatozoa (P = 0.1474); no viability differences were noted for fresh spermatozoa (P ⩾ 0.2298). The cryopreservation process did not affect acrosomal morphology of the indo-1-exposed spermatozoa (P ⩾ 0.1147). These data indicate that Ca2+ regulation by Ca2+- and A23187-challenged bull spermatozoa differs following cryopreservation procedures, possibly relating to the reduced fertilization capacity of commercially cryopreserved semen.

Survival of Bull Spermatozoa Seeded and Frozen at Different Rates in Egg Yolk-Tris and Whole Milk Extenders

Six factorially arranged experiments were designed to study effects of seeding, freezing, and thawing rates in whole milk and egg yolk-Tris extenders commonly used for commercial cryopreservation of bull sperm. In these extenders, semen normally is supercooled to –13 or –14°C unless the sperm are seeded. When sperm were supercooled or seeded, either mechanically or with immobilized silver iodide, and frozen to –196°C, the postthaw percentages of motile sperm were 59, 57, and 64%, respectively. Freezing rates of –15, –25, and –35°C/min gave similar sperm survival rates and were superior to –5°C/min. For milk, the critical freezing temperature extended to –75°C before transfer to liquid nitrogen gave good results. For egg yolk-Tris extender, transfer to liquid nitrogen was less critical once –50°C had been attained. Thawing of sperm in water baths at 25 and 45°C gave similar results, and both temperatures were superior to 5°C. The postthaw percentage of motile sperm in egg yolk-Tris was equal or superior to that of sperm frozen in milk. A freezing rate of –15°C/min to –100°C and thawing at 25°C consistently gave good results.