Percentage Of Intact Acrosomes Research Articles

Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa

BackgroundProximal cytoplasmic droplets (PCDs), a remnant of germ cell cytoplasm, are common non-specific morphological defects in bovine semen. This study evaluated the effect of higher percentages of PCDs on the quality of frozen-thawed bovine semen, embryo production and early embryo development.MethodsThree ejaculates from each of five (group 1: PCD ≤ 1%, control) and eight adult Bos indicus bulls (group 2: PCD ≥ 24%) were analysed. Semen samples were examined for: post-thaw motility, vigour of movement, concentration, sperm morphology, slow thermoresistance test (STT), membrane integrity, acrosome status, mitochondrial function using fluorescent probes association (FITC-PSA, PI and JC-1) and sperm chromatin integrity using acridine orange assay. Two bulls from group 2, with 28.5% and 48.5% PCD, respectively, and three bulls from the control group, each with 0% PCD, were selected for IVF (in vitro fertilisation).ResultsSemen analyses revealed a significant correlation (P < 0.01) between increased rates of PCD and sperm quality traits. Nevertheless, no differences were observed in sperm motility and vigour either before or after the STT or in the percentage of intact acrosomes (analysed by differential interference contrast microscopy (DIC) after STT), but membrane integrity, acrosome status (evaluated with FITC-PSA staining method after thawing) and mitochondrial function were reduced, when compared with group 1 (P < 0.05). The higher incidence of PCD was positively correlated to chromatin damage, especially after three hours of incubation at 37°C. IVF showed similar results for bull C2 (group 1, control) and bull P2 (group 2, group with higher PCDs).ConclusionHigher PCD levels influenced spermatozoa quality traits. IVF and embryo development data showed that cleavage, blastocyst formation and blastocyst hatching may have been influenced by the interaction of morphology traits and individual bull effects.

Effects of Egg Yolk Source on the Cryopreservation of Stallion Spermatozoa

Three experiments were conducted to determine whether replacement of chicken egg yolk, as a component of freezing extenders, with egg yolk from other avian species would improve the post-thaw motility and percentage of intact acrosomes of stallion spermatozoa. In the first experiment, substitution of chicken egg yolk with chukar egg yolk, as a component of the lactose–ethylenediaminetetraacetic acid extender, improved (P ≤ .05) the post-thaw motility of stallion spermatozoa. These results were not replicated in (IMV Technologies, Maple Grove, MN, USA) a more expansive study comparing 2%, 4%, 6%, or 8% egg yolk combined with INRA 96 when a “slow freeze” method was used, or the same substitution at levels ranging from 13% to 22% when egg yolk was combined with lactose–ethylenediaminetetraacetic acid for diluents used for a “fast freeze” method of cryopreservation. In the third study, egg yolks from regular and high omega-3 chicken eggs as well as from turkey, chukar, and mallard duck eggs were analyzed for lipid content and fatty acid profile. The yolk from the turkey eggs was higher (1,300 mg/100 g) and that from mallard ducks was lower (560 mg/100 g) in cholesterol as compared with the two types of chicken eggs and chukar egg yolk (range, 1,046-1,094 mg/100 g). In addition, the high omega-3 eggs did test higher for fatty acids (4.51 g/100 g) than other types of eggs (range, 0.28-0.73 g/100 g). Substitution of chicken egg yolk with turkey, but not duck, egg yolk resulted in higher post-thaw total motility (P ≤ .05) for spermatozoa obtained from two of the three stallions used in the third experiment.

Effect of storage of domestic cat ( Felis catus) epididymides at 5 °C on sperm quality and cryopreservation

Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat ( Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91 ± 2.02, 48.21 ± 1.47, and 43.03 ± 1.32) and after thawing (51.81 ± 3.02, 41.90 ± 2.14, and 42.35 ± 1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33 ± 1.38% before and 52.50 ± 1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22 ± 1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI = 39.17 ± 2.76 and 45.00 ± 2.65, respectively) and the percentage of intact acrosomes (45.76 ± 4.91% and 60.67 ± 3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24 h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.

Effect of Centrifugation Technique on Post-storage Characteristics of Stallion Spermatozoa

Three ejaculates were collected from four stallions and used to compare the effects of three centrifugation methods on post-storage motility and recovery of available sperm. Two aliquots per ejaculate were diluted with skim milk-glucose (SKMG) extender to 50×10 6 sperm/mL, placed in 50-mL conical bottom tubes, and centrifuged at either 700 g for 15 minutes (700 g) or 600 g for 12 minutes (600 g). A third aliquot was diluted 1:1 with SKMG, placed in 15-mL conical tubes, and centrifuged at 400 g for 7 minutes (400 g). Subsamples from each pre-treated diluted ejaculate were held at room temperature and evaluated for motility at the same time as the post-centrifugation pre-storage motility evaluation was made for treated aliquots. After centrifugation, samples from each aliquot were stored at 5°C for evaluation after 24 and 48 hours or frozen in liquid nitrogen. Percentage of available sperm harvested was higher ( P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400 g versus 600 g in 50-mL tubes. After centrifugation, total but not progressive motility of aliquots centrifuged at 700 g was lower than that for noncentrifuged controls and sperm from aliquots centrifuged at 400 g in 15-mL tubes. After cold storage, values for total but not progressive motility or velocity were higher ( P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400 g compared with those centrifuged in 50-mL tubes at both 600 g and 700 g. Postthaw motility of frozen sperm was not different between centrifugation treatments. Poststorage percentages of intact acrosomes and detached heads did not differ because of centrifugation treatment.

Improved Quality of Cryopreserved Cheetah (Acinonyx jubatus) Spermatozoa After Centrifugation Through Accudenz

Sperm cryopreservation, in combination with assisted reproductive techniques, is a valuable tool for the genetic management of endangered felids. However, the acrosome of the cheetah spermatozoon is especially sensitive to cryopreservation, with approximately 40% of spermatozoa experiencing acrosomal damage immediately after thawing and then another approximately 15% loss during the next 4 hours in vitro. Additionally, thawing causes a reduction in sperm motility by approximately 20% with another decrease of approximately 12% during subsequent incubation in vitro. We hypothesized that slow removal of glycerol from cryopreserved cheetah spermatozoa using an Accudenz gradient would improve acrosomal integrity, sperm motility longevity, and structural morphology. Accudenz was compared with traditional cheetah sperm processing methods for glycerol removal that involves washing, multistep resuspension, and swim-up processing. Electroejaculates (n = 21 total from 8 males) were washed in Ham F10 medium, and sperm pellets were resuspended in TEST-yolk buffer with 0% glycerol. Samples were cryopreserved in straws in 4% final glycerol, thawed, and assessed for percent intact acrosomes (% IA), percent motility (% M), and forward progressive status (FPS; scale, 0-5). Sperm motility index (SMI) was calculated as (% M + [FPS x 20]) / 2. In study 1, glycerol removal by centrifugation through an Accudenz gradient (4%, 10%) was compared with traditional sperm washing (control) and multistep resuspension protocols. At each time after centrifugation (hourly for 4 hours), % IA was improved (P < .05) for Accudenz (range, 36%-39%) compared with control (30%-33%) and multistep (29%-33%) treatments. In study 2, a modified Accudenz protocol was compared with traditional washing and was found to improve (P < .05) SMI (range, 52-64) compared with controls (range, 41-52) at each time postthaw after centrifugation. In study 3, swim-up processed sperm were compared with those treated by centrifugation through Accudenz and traditional sperm washing for improving sperm morphology. The percentage of structurally-normal sperm recovered postthawing increased (P < .05) for both the Accudenz (38%) and swim-up (33%) treatments compared with controls (21%). Percent IA and SMI also were improved (P < .05) for Accudenz (range, 39%-47% and 46-59, respectively) compared with controls (range, 26%-33% and 40-53, respectively). Results indicate that using Accudenz for glycerol removal from cryopreserved cheetah sperm mitigates the significant loss in sperm quality that occurs after freeze-thawing. This alleviation of cellular damage resulting from cryopreservation contributes to a more than 10% improvement in overall sperm motility and, more importantly, allows retention of 40% or more of sperm with intact acrosomes.

The Effect of Different Concentrations of Glycerol and DMSO on Viability of Markhoz Goat Spermatozoa During Different Freezing Temperatures Steps

The present study was conducted to determine the following; (1) the influence of different concentrations of glycerol (1, 3, 5 or 7%, v/v) in experiment 1 and DMSO (1, 1.25, 1.5 or 1.75% v/v) in experiment 2 added either at 37 or 5 degrees C and (2) the comparing of best concentration of glycerol with the best of DMSO, obtained in this study, on post-thaw motility, progressive motility, viability and normal acrosome of Markhoz goat sperm. In experiment 1, motility, progressive motility and viability of sperm were improved significantly (p < 0.05) by increasing of glycerol concentrations in the extenders, with the best results obtained with glycerol at 7% added at 37 degrees C. However, the rate of normal acrosome showed an opposite trend, i.e., the extender containing 1% glycerol added at 5 degrees C showed better results (p < 0.05). In experiment 2, the observed results showed similar tendencies to experiment 1. The data showed that the extender containing 1.75% DMSO concentration (the highest level) added at 37 degrees C was significantly (p < 0.05) better than others. The percentage of intact acrosomes decreased significantly (p < 0.05) by increasing of DMSO concentrations, when added at 37 degrees C. Further, the results of 1% DMSO added at 5 degrees C was (p < 0.05) better than other groups. In regard to all evaluated parameters, the observed results in experiment 3 showed that extender containing 7% glycerol added at 37 degrees C was significantly (p < 0.05) better than 7% glycerol added at 5 degrees C and extender containing 1.75% DMSO added at both temperatures. In conclusion, the results of presented study indicated that glycerol is still the cryoprotectant of choice for freezing of Markhoz goat sperm.

Assessment of semen quality, sperm cryopreservation and heterologous IVF in the critically endangered Iberian lynx (Lynx pardinus)

Semen traits and factors affecting sperm cryopreservation were assessed in the Iberian lynx (Lynx pardinus), a species regarded as the most endangered felid in the world. For cryopreservation, semen was washed, resuspended in a Tes-Tris-based diluent (TEST) or a Tris-based diluent (Biladyl), both with 20% egg yolk and 4% glycerol, loaded into straws, cooled to 5 degrees C using an automated programmable system and frozen on nitrogen vapour. Heterologous IVF of in vitro-matured domestic cat oocytes was used to test the fertilising ability of cryopreserved spermatozoa. Electroejaculates from five males were obtained. Characterisation of the electroejaculates revealed mean (+/- s.e.m.) values of 3.3 +/- 0.6 x 10(6) total spermatozoa, 73.6 +/- 4.6% motile spermatozoa, 23.7 +/- 4.0% morphologically normal spermatozoa and 40.7 +/- 2.3% spermatozoa with intact acrosomes. After thawing a higher percentage of motile spermatozoa was seen in TEST than in Biladyl (34.0 +/- 6.2% v. 7.5 +/- 4.8%, respectively; P < 0.05); however, there were no differences in the percentage of intact acrosomes between the two diluents. Iberian lynx spermatozoa fertilised domestic cat oocytes in vitro, with higher fertilisation rates observed for spermatozoa cryopreserved in TEST than in Biladyl, although the difference did not reach statistical significance (20.5 +/- 4.5% v. 11.5 +/- 6.8%, respectively). There were positive significant relations between the fertilisation rates and both the percentage of normal spermatozoa and the percentage of spermatozoa with an intact acrosome before cryopreservation (P = 0.04). This first report of the collection and cryopreservation of Iberian lynx semen and analysis of fertilising ability is an important step in the development of assisted reproductive techniques for this critically endangered felid species.

Lead chloride affects sperm motility and acrosome reaction in mice

Lead is highly toxic and persistent in the environment and, thus, a major concern for public health. In this study, the effects of lead chloride (PbCl2) on mouse epididymal sperm were evaluated. Male mice were subcutaneously injected with 74 and 100 mg PbCl2/kg body weight for four consecutive days. Sperm was collected from the epididymis and several parameters of sperm function, such as sperm density, motility, viability, mitochondrial function, acrosome integrity and morphology, were evaluated. Furthermore, DNA fragmentation was assessed by the terminal deoxylnucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay and chromatin integrity was evaluated by sperm chromatin structure assay (SCSA). In order to assess direct effects on existing sperm population, we sacrificed one group for each condition at day 5. The effects of lead upon one entire spermatogenic cycle were evaluated on day 35. Both lead concentrations used in this work affected sperm motility, although no significant differences were observed in sperm viability, mitochondrial function and DNA/chromatin integrity. However, a decrease in the percentage of intact acrosomes was also observed, mirroring a lead-induced premature acrosome reaction. Thus, the results obtained indicate that, together with impaired motility, the effect of lead toxicity on acrosome integrity, leading to premature reaction, may compromise the ability of sperm to fertilize the oocyte.

Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation

The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS-PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen-thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P<0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P<0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity.

Preservation of boar semen at 18 °C induces lipid peroxidation and apoptosis like changes in spermatozoa

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (Δ Ψ m) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 °C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, Δ Ψ m, and membrane permeability. The lipid peroxidation status of the sperm was assessed based on the malonaldehyde (MDA) levels. Detection of Δ Ψ m was done using 3,3′-dihexyloxacarbocyanine iodide [DiOC 6(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly ( p < 0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/10 9 sperm) were low in sperm (99.83 ± 2.69) and seminal plasma (191.98 ± 11.58), which gradually increased up to the 96 h of storage. Highest negative correlation ( r value) was observed between MDA levels and sperm motility (−0.97), live percent (−0.97), acrosomal integrity (−0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (−0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage ( r = 0.98). There was a significant ( p < 0.05) increase in the sperm cells with low Δ Ψ m from 0 to 96 h of preservation. Before preservation, 14.85 ± 4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00 ± 6.25%. The apoptotic sperm population was 8.33 ± 2.31% in fresh semen, while this population was 25.19 ± 4.25% at 96 h of preservation and the difference was significant ( p < 0.05). The findings of the present study revealed that liquid preservation of boar semen at 18 °C induces lipid peroxidation, decrease mitochondrial membrane potential and increase the plasma membrane permeability.

Electroejaculation and semen characteristics of Asiatic Black bears ( Ursus thibetanus)

This study details the seminal traits in the rare Asiatic black bear, revealing that this species generally produces high quality, concentrated ejaculates containing sperm of high motility and morphological integrity and similar to other members of the Ursine lineage. Semen was collected by electroejaculation and 23 trials were performed on 18 bears. The mean values were obtained for volume (0.61 ml), pH (7.1), sperm concentration (1049 × 10 6 ml −1), total sperm counts (502.8 × 10 6), motility percentage (63.8%), forward progressive status (3.5), abnormal sperm percentage (37.9%) and intact acrosome percentage (76.6%).

ASSESSMENT OF VIRAL PRESENCE IN SEMEN AND REPRODUCTIVE FUNCTION OF FROZEN-THAWED SPERMATOZOA FROM PALLAS' CATS (OTOCOLOBUS MANUL) INFECTED WITH FELINE HERPESVIRUS

Although herpesviruses are known to contaminate the semen of several mammalian species, the occurrence of feline herpesvirus type 1 (FHV-1) in semen of infected cats has not been reported. Our objectives in this study were to investigate the presence of FHV-1 DNA in seminal fluid and frozen-thawed spermatozoa from FHV-1 infected Pallas' cats (Otocolobus manul) and assess the functionality of their frozen-thawed spermatozoa in vitro. Over a 3-yr period, semen (n = 33 ejaculates) was collected periodically via electroejaculation from four Pallas' cats chronically infected with FHV-1. Spermic ejaculates were frozen by pelleting on dry ice and stored in liquid nitrogen. After thawing, sperm motility and acrosome status were assessed over time during in vitro culture. For vitro fertilization (IVF), viable domestic cat (Felis silvestris catus) oocytes were inseminated with frozen-thawed Pallas' cat spermatozoa and evaluated for embryo cleavage. For FHV-1 polymerase chain reaction (PCR) analysis, DNA was extracted from seminal fluid, frozen-thawed spermatozoa, inseminated oocytes, heterologous IVF embryos, and conjunctival biopsies and analyzed for presence of a 322-base pair region of the FHV-1 thymidine kinase gene. Immediately post-thaw, sperm motility and percentage of intact acrosomes were decreased (P < 0.05) compared to fresh samples, and declined further (P < 0.05) during culture. However, all frozen-thawed IVF samples were capable of fertilizing domestic cat oocytes (overall, 46.1 +/- 6.0% cleavage). PCR analysis did not identify FHV-1 DNA in any reproductive sample despite the repeated detection of FHV-1 DNA in conjunctival biopsies. These results suggest that semen collected from Pallas' cats infected with FHV-1 does not contain cell-associated or non-cell-associated virus and that frozen-thawed spermatozoa exhibit adequate function for potential genetic rescue with minimal risk of FHV-1 transmission.

O efeito das enzimas hialuronidase e tripsina na liquefação do sêmen de macacos pregos (Cebus apella)

Os efeitos das enzimas hialuronidase e tripsina foram avaliados quanto à liquefação, motilidade, vigor e integridade do acrossoma, no sêmen de seis macacos pregos (Cebus apella), mantidos na Fundação Parque Zoológico de São Paulo. O sêmen foi colhido por eletroejaculação, após anestesia geral, e a fração líquida foi imediatamente avaliada. A fração coagulada do sêmen foi tratada com as enzimas hialuronidase e tripsina, na dose de 1mg/ml, diluídas em meio 199 (Nutricel, Campinas/SP, Brasil), na proporção 1:4 e examinada após 5 e 15 minutos. Test t de Student foi utilizado para comparar os tratamentos. Não houve diferença significativa quanto a motilidade, vigor e integridade de acrossoma (p &gt;; 0.05), entre a fração de sêmen coagulado diluído em hialuronidase e tripsina, após cinco ou quinze minutos. No entanto, houve diferença significativa quanto a motilidade e vigor entre a fração líquida e a coagulada do sêmen (p &lt; 0.05), após quinze minutos. Não houve diferença significativa com relação à integridade de acrossoma (p &gt;; 0.05) entre a fração líquida e coagulada do sêmen, após 15 minutos. De acordo com os resultados, podemos concluir que não houve efeito aparente na fração coagulada do sêmen tratado com as enzimas hialuronidase e tripsina com relação a motilidade, vigor e integridade do acrossoma. No entanto, houve diferença significativa entre a fração líquida e coagulada do sêmen com relação a motilidade e vigor, porém não quanto à integridade do acrossoma. De maneira geral, em ambos os tratamentos, não houve a completa dissolução do coágulo.

Influence of cooling rates and addition of Equex pasta on cooled and frozen-thawed semen of generic gray ( Canis lupus) and Mexican gray wolves ( C. l. baileyi)

A current priority for the preservation of the endangered Mexican gray wolf ( Canis lupus baileyi) is the development of a sperm-based genome resource bank for subsequent use in artificial insemination. To optimize the quality of cryopreserved sperm, the procedures involved in processing semen before and during freezing need to be improved. The aim of this study were to examine the effects of: (i) different cooling periods before freezing and (ii) addition of Equex pasta (Minitüb, Tübingen, Germany) on the characteristics of sperm from the generic gray wolf and the Mexican gray wolf after cooling and cryopreservation. For Mexican wolf sperm, cooling for 0.5 and 1.0 h had a less detrimental effect on cell morphology than cooling for 2.5 h, whereas the slower cooling rate (2.5 h) had a less detrimental effect on functional parameters and seemed to cause less damage to plasma membrane and acrosome integrity than 0.5 and 1.0 h. For the generic gray wolf, cooling semen for 2.5 h had less detrimental effect on plasma membrane integrity and viability; together with the 0.5 h cooling time, it yielded the highest percentages of intact acrosomes. As previously shown in the domestic dog, Equex pasta had no beneficial effect on sperm characteristics in either wolf species.

Characteristics of Frozen-Thawed Spermatozoa Cryopreserved with Different Concentrations of Glycerol in Captive Japanese Black Bears (Ursus thibetanus japonicus)

Seven mature Japanese black bears were used as semen donors, and a total of 7 semen samples collected from the animals by the electroejaculation method were cryopreserved in liquid nitrogen. Egg yolk-TRIS-citrate-glucose extender was used, and the effects of different final concentrations of glycerol, at 4-12% (v/v), on frozen-thawed spermatozoa were examined. No significant difference was observed in percent motility or percent abnormal morphology of frozen-thawed spermatozoa among the different glycerol concentrations. Percent viability and percent intact acrosomes of spermatozoa cryopreserved with 4 and 6% glycerol were significantly higher than those with 10 and 12% glycerol. These results suggest that a suitable glycerol concentration for freezing Japanese black bear semen within the range tested would be 4-6%.

Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa

Four experiments were conducted to investigate the effects of egg yolk during the freezing step of cryopreservation (namely, the process except for the cooling step), on the viability of goat spermatozoa. The effects of egg yolk on sperm motility and acrosome integrity during the freezing step were investigated in Experiment 1. Spermatozoa diluted with Tris-citric acid-glucose (TCG) solution containing 20% (v/v) egg yolk were cooled to 5 °C, washed, and then frozen in TCG with egg yolk (TCG-Y), TCG without egg yolk (TGG-NY), 0.370 M trehalose with egg yolk (TH-Y), or trehalose without egg yolk (TH-NY). All extenders contained glycerol. In frozen-thawed spermatozoa, the inclusion of egg yolk in the freezing extenders increased ( P<0.05) percentages of motile sperm, progressively motile sperm, and the recovery rate (ratio of post-thaw to pre-freeze values), but decreased ( P<0.05) acrosomal integrity. Moreover, extenders with trehalose had better ( P<0.05) post-thaw sperm viability. In Experiment 2, the effects of egg yolk on acrosome status before and after freezing were studied. Egg yolk significantly decreased the proportion of intact acrosomes before freezing, leading to fewer ( P<0.05) intact acrosomes post-thaw and lower ( P<0.05) recovery rates for intact acrosomes. In Experiment 3, including sodium dodecyl sulfate (SDS) in a diluent containing egg yolk tended to preserve the acrosome compared with the egg yolk containing diluent free of SDS, however, spermatozoa had a lower ( P<0.05) proportion of intact acrosomes than those in a yolk-free diluent. However, after cooling, spermatozoa were diluted with a glycerolated extender containing egg yolk. Therefore, the objective of Experiment 4 was to explore whether the egg yolk or glycerol was responsible for the reduced intact acrosome percentage. In this experiment, after cooling and washing the spermatozoa were diluted in TCG with glycerol and/or egg yolk. The combination of glycerol and egg yolk in the extender reduced ( P<0.05) the proportion of intact acrosomes compared with egg yolk or glycerol alone. In conclusion, the inclusion of egg yolk significantly improved sperm motility, indicating its beneficial effects during the freezing step of cryopreservation; trehalose appeared to synergistically increase its cryoprotective effects. Furthermore, although neither glycerol nor egg yolk per se affected the proportion of intact acrosomes, the combination of the two significantly reduced the proportion of acrosome-intact spermatozoa.

The partial head decondensation test is a new, quick method to assess acrosome status in human spermatozoa

Objective To develop a fast method for assessing acrosome status in human spermatozoa. Design Development of a new in vitro test to assess acrosome reaction in human spermatozoa. Setting Academic medical institution. Patient(s) Normozoospermic subjects. Intervention(s) Spermatozoa were isolated from fresh semen samples, capacitated, and stimulated or not with P or ionomycin. Acrosome reactions were evaluated by phase-contrast microscopy after a brief sperm incubation in a decondensing solution. The results were compared with those obtained by scanning electron microscopy and fluoresceinated lectin staining. Main outcome measure(s) Percentage of intact acrosomes. Result(s) The new procedure allowed intact acrosomes to be easily identified and quantified by phase-contrast microscopy. In unstimulated and ionomycin-treated spermatozoa, a very good agreement was found among the new test, scanning electron microscopy, and fluoresceinated lectin staining. In P-treated spermatozoa, the proposed method allowed a significantly higher percentage of reacted acrosomes to be resolved, likely due to its ability to detect the very initial stages of the acrosome reaction. Conclusion(s) The new test allows acrosome-intact and acrosome-reacted spermatozoa to be unambiguously singled out and quantified. The method is rapid, reliable, sensitive, and easy to perform, which makes it of profitable use in both basic research and diagnostic practice.

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 °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 6 h 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 °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.

Functional evaluation of dog ejaculates with priority given to the aspect of acrosome integrity

38 ejaculates of dogs of various breeds from2to 6 years old were examined in a short-term 120 min survival test. The examination focused on the functional parameters of sperm (motility, percentage of live sperma&amp;shy;tozoa, acrosomal integrity) and their morphology. The levels of relationship between individual criteria of sperm analysis were demonstrated. Statistically highly significant correlations between motility, ratio of live spermatozoa, percentage of intact acrosomes and percentage of spermatozoa with altered superficial structures were proven. Our findings proved the sensitivity of superficial structures, primarily plasmatic membranes, and the higher resistance of structures with an organelle basis. Strict morphological sperm analysis provides important information about the rise in qualitative alterations of ejaculates caused by changes in superficial structures during the course of a short-term survival test.

The effect of nocturnal sampling on semen quality and the efficiency of collection in bovine species

This study evaluated night and day semen collection regimes in Holstein and Brahman bulls (four bulls of each breed) that were collected weekly, each during a morning and a night collection. Ejaculates ( n=64) were obtained via artificial vagina over 4 weeks. The first collection of each week alternated between night and day. Two collection teams were employed. Bull behavior parameters included reaction time to first mount, time to ejaculation, a refractory period test, and a thrust intensity test. The numbers of interruptions were counted as a managerial parameter. Pre-freeze semen parameters included total volume, initial motility and concentration. Post-freeze semen parameters measured were: 0- and 3-h post-thaw motility; percent intact acrosomes; and percent sperm abnormalities. Data were analyzed by least squares methods. The bull within breed effect differed ( P<0.05) for behavior parameters. The bull within breed effect for total motile sperm harvested was not significant. The bull within breed response was mixed for post-freeze semen viability parameters. Bull within breed was not significant for sperm abnormalities. The night versus day treatment was significant for the managerial parameter ( P=0.002). Although a different collection schedule for Bos indicus cattle was not warranted, the efficiency of the collection process was affected by extraneous environmental conditions.