Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation

In this paper, DNA laddering analysis and single-cell gel electrophoresis (SCGE) or Comet assay, were used to detect DNA damage in response to a cryopreservation process in sea bass spermatozoa. The results obtained demonstrate that the cryopreservation protocol used to cryopreserve the sea bass sperm cause significantly damage at DNA level. In fact, the degree of DNA damage in frozen-thawed sperm (%DNAT=38.2+/-11.2, MT=498.9+/-166.4, n=3) was different (P<0.01) from that measured in fresh sperm (%DNAT=32.7+/-11.1, MT=375.2+/-190.7, n=3). Data here reported also demonstrated the fundamental role played by cryoprotectants (BSA and Me2SO) in reducing fish sperm DNA fragmentation. Finally, from our results, the ability of SCGE to reveal DNA fragmentation in fish sperm is also confirmed.     

DNA integrity of fresh and frozen canine epididymal spermatozoa

The aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1 mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3 ± 3.6) and samples frozen with (4.2 ± 3.8) or without (3.6 ± 3.7) melatonin. Sperm motility was significantly (p < 0.0001) higher in fresh compared to frozen samples. The presence of melatonin in the freezing extender did not affect the sperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (p < 0.01) by cryopreservation, and melatonin did not exert any beneficial effects. In conclusion, DNA fragmentation of canine epididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques.     

Effects of different cryoprotective agents on ram sperm morphology and DNAintegrity

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

Impact of Eurycoma longifolia extract on DNA integrity,lipid peroxidation,and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.     

Comparison of sperm quality and DNA integrity in mouse sperm exposed to various cooling velocities and osmotic stress

The first objective was to compare sperm quality following conventional manual sperm freezing (cryovials held 1, 2, 3, and 4 cm, respectively, above liquid nitrogen (LN₂) for 10 min, resulting in cooling velocities of approximately −14.9, −10.1, −6.6, and −5.1 °C/min, respectively), and cooling velocities of −5, −20, −40, and −100 °C/min in a programmed automated freezer, for sperm recovered from CD-1, B6129SF1, and C57BL/6NCrlBR mice. Furthermore, using these strains, as well as 129S/SvPaslco, and DBA/2NCrlBR mice, the second objective was to determine the effects on DNA integrity of sperm exposed to hyposmotic (1 mOsm/L) and hyperosmotic (2400 mOsm/L) solutions, compared to an isosmotic control (300 mOsm/L). For freezing above LN₂ or in an automated freezer, 2 cm above LN₂ and −100 °C/min, respectively, were optimal (P < 0.05-0.01), with no significant differences between these two approaches for post-thaw progressive motility, DNA integrity, and in vitro rates of fertilization and blastocyst formation. Both manual and automated freezing techniques increased post-thaw sperm DNA fragmentation (P < 0.01); the DNA integrity of post-thaw sperm was significantly affected by cooling velocity and strain background. Relative to isosmotic controls, a hyposmotic solution was more deleterious (P < 0.05-0.01) to sperm DNA integrity than a hyperosmotic solution for CD-1, B6129SF1, C57BL/6, and DBA mice (there were strain-dependent differences). In conclusion, optimization of freezing distance and cooling velocity (manual and automated freezing, respectively) were significant factors for efficient cryopreservation and re-derivation of mice from frozen-thawed sperm. Additionally, osmotically-driven volume changes in mouse sperm increased DNA fragmentation, with susceptibility affected by background strain.     

Effects of different concentration and combinations of cryoprotectants on sperm quality,functional integrity in three Indian horse breeds

Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acrosome and DNA integrity as well as other post thaw seminal characteristics of semen of three Indigenous stallion breeds. In the current study, semen was collected from apparently healthy 4–6 years old 3 Marwari, 3 Manipuri and 3 Zanskari breed stallions. After semen collection and evaluation of fresh semen, each semen sample was extended with semen extender containing different concentrations and combinations of Gly and DMF cryoprotectants (i.e. 5% Gly, 5% DMF, 2% Gly, 2% DMF, 2.5% Gly +2.5% DMF and 1% Gly +1% DMF) and frozen. Post thaw semen evaluation was done on the basis of post thaw motility, live sperm count, hypo osmotic swelling test, acrosomal integrity and DNA integrity. Frozen thawed semen showed that the values of plasma membrane integrity, acrosome integrity and DNA integrity parameters were significantly higher (P < 0.05) with 5% DMF than the other cryoprotectants levels and combinations of Gly and DMF. From the present study, it was inferred that the combination of cryoprotectants at different concentrations (Gly and DMF @ 2.5 and 1%) also could not show better enhancement compared to the single cryoprotectant i.e DMF @5% in various post thaw seminal characteristics of Indigenous stallion semen. DMF at 5% concentration gave better protection to the plasma membrane and retained the acrosome and DNA integrity of the spermatozoa. Hence it can be concluded that DMF at 5% can be used for the cryopreservation of the Indigenous stallion with better preservation of the seminal quality.     

Fresh and frozen-thawed sperm quality, nuclear DNA integrity, invitro fertility, embryo development, and live-born offspring of N-ethyl-N-nitrosourea (ENU) mice

Efficient collection, freezing, reliable archiving of sperm, and re-derivation of mutant mice are essential components for large-scale mutagenesis programs in the mouse. Induced mutations (i.e. transgenes, targeted mutations, chemically induced mutations) in mice may cause inherited or temporary sterility, increase abnormal sperm values, or decrease fertility. One purpose of this study was to compare the effect(s) on fresh and frozen-thawed sperm quality, spermatozoa DNA integrity, unassisted in vitro fertility (IVF) rate, in vitro embryo development rate to blastocysts, and live-born offspring rates in non-ENU (control) animals and the F1-generation of N-ethyl-N-nitrosourea (ENU)-treated male mice (765 mg/kg C57BL6/J or 600 mg/kg 129S1/SvImJ total dose). The second purpose was to determine the effect(s) of parental oocyte donor strain on in vitro fertilization, in vitro embryo development to blastocysts, and live-born offspring rates using sperm and unassisted IVF to re-derive animals from non-ENU control and ENU mice. Sperm assessment parameters included progressive motility, concentration, plasma membrane integrity, membrane function integrity, acrosome integrity, and DNA integrity. There were no significant differences in fresh sperm assessment parameters, DNA integrity, unassisted in vitro fertility rate, in vitro embryo development rate to blastocysts, and live-born offspring rates between non-ENU and C3B6F1/J or B6129S1F1/J ENU mice. In addition, there were no significant differences in frozen-thawed sperm assessment parameters and DNA integrity rates for non-ENU control and ENU C3B6F1/J or B6129SF1/J mice. In vitro fertilization and in vitro embryo development to blastocysts were effected from strain genetic variability (P < 0.05). However, the cryopreservation process caused an increase of DNA fragmentation in non-ENU control and ENU C3B6F1/J or B6129S1F1/J hybrid mice compared to fresh control sperm (P < 0.01). Unlike the combinations of hybrid sperm and hybrid oocyte, increasing frozen-thawed sperm DNA fragmentation decreased the embryo development rate to blastocyst compared to fresh sperm when C57BL6, C3H, or 129S inbred mice were used as oocyte donors (P < 0.05).     

Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice

Evidence exists to suggest detrimental effects of heat stress on male fertility. This study was designed to assess the effects of scrotal heat stress on mature and developing sperm in a mouse model. After receiving shock heat treatment (42 degrees C for 30 min), mature spermatozoa were recovered from the epididymis hours (6) or Days (7, 14, 21, 28, 60) later, to determine the variables: number of spermatozoa, sperm viability, motility and progressive motility, sperm DNA integrity as established by the TUNEL method, embryo implantation rate, and sex ratio of the fetuses conceived using the heat-exposed spermatozoa. Our results indicate that transient mild heat treatment does not affect in the same way the different types of male germ cells. Spermatocytes present within the testis at the time of heat stress resulted into a lower concentration of spermatozoa with reduced viability and low motility. Even though, DNA integrity of spermatozoa resulting from spermatocytes was also compromised by heat stress, the higher degree of DNA damage was found among spermatozoa resulting from spermatids present within the testis at the time of heat stress. At last, heat shock effect on spermatozoa present in the epididymis at the time of thermal stress resulted into a sex ratio distortion. These findings point to a higher sensitivity of spermatocytes to heat exposure and also suggest a different response of X and Y chromosome-bearing spermatozoa to heat stress that warrants further investigation.     

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

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

The advantages of LDL (low density lipoproteins) in the cryopreservation of canine semen

A medium containing LDL (Low Density Lipoproteins, the cryoprotective component of chicken egg yolk) was compared with egg yolk for the preservation canine spermatozoa during the freeze–thaw process. Twenty sperm samples taken from 10 dogs were frozen in liquid nitrogen at −196 °C in seven different media: one control medium containing 20% egg yolk, and six test media containing 4%, 5%, 6%, 7%, 8%, and 10% LDL, respectively.Following thawing, sperm motility was assessed using a Hamilton-Thorne Sperm Analyser equipped with the CEROS 12 software. The percentage of motile spermatozoa was 55.3% in the 6% LDL medium (optimal concentration) compared with 27.7% in the egg yolk based medium (p < 0.05).In comparison with the egg-yolk medium, the LDL medium also resulted in an improved preservation of spermatozoa during the freezing process (p < 0.05) in terms of acrosomal integrity (FITC-PSA test), flagellar plasma membrane integrity (HOS test), and DNA integrity (Acridine Orange test).In addition, six Beagle bitches were inseminated twice, via the intra-uterine route, at an interval of 24 h; 200 × 10⁶ spermatozoa that had been previously frozen in the 6% LDL medium were used per insemination. All of the bitches became pregnant (gestation rate of 100%).In conclusion, the 6% LDL medium provides improved protection of the spermatozoa during the freeze–thaw process and a marked improvement in the motility parameters of canine spermatozoa in comparison with the control medium containing egg yolk alone.Finally, the use of LDL as a cryoprotectant for canine semen does not interfere with fertility.     

Pentoxifylline increase sperm motility in devitrified spermatozoa from asthenozoospermic patient without damage chromatin and DNA integrity

The freeze–thaw process results in reduced motility, viability and fertilization potential of human spermatozoa. So, a variety of substances were evaluated in order to enhance human sperm resistance to the stress of cryopreservation, such as Pentoxifylline (PTX) for improving the Intracytoplasmic sperm injection (ICSI) outcomes. The aim was to investigate the effect of PTX on sperm parameters and chromatin/DNA integrity of asthenozoospermic semen post vitrification. A total of 30 semen specimens were obtained from infertile men with asthenozoospermia. The cryoprotectant-free vitrification was performed for the samples after assessment of sperm parameters. After warming, each sample was exposed for 30 min to 3.6 mmol/l PTX in experimental group and the control group without any treatment apposing at 37 °C for 30 min in regard, to repeat all in vitro analysis (sperm parameters and DNA integrity assay). Regardless of the vitrification devastating impacts on sperm parameters, incubation of post vitrified samples with PTX increased the rate of progressive motility (P < 0.01). Moreover, PTX addition did not significantly damage DNA integrity of asthenozoospermic sperm samples. The data showed that PTX was able to improve sperm movement without any adverse effects on sperm chromatin/DNA integrity in vitrification program.     

Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm

Assisted reproduction using frozen-thawed semen has practical advantages, although cryopreservation is detrimental to sperm fertility in most mammals. We examined the influence of cryopreservation and reactive oxygen species (ROS) on ram sperm DNA stability (using SCSA), lipid peroxidation (LPO), chlortetracycline fluorescence (CTC) patterns, motility and viability. In Experiment 1, DNA integrity, LPO, CTC, motility and viability tests were performed on fresh and cryopreserved sperm after 0, 6, and 24 hr in synthetic oviductal fluid (SOF). In Experiment 2, fresh sperm were incubated in serum-free SOF (SOF-S; 1, 4, and 24 hr) with 0, 50, 150, or 300 microM H2O2 then assayed. Cryopreservation increased the percentage of sperm with a high DNA fragmentation index (%DFI), decreased the percentages of motile and viable sperm at thawing (0 hr), but did not affect LPO. H2O2 (150 or 300 microM) increased %DFI after 24 hr. LPO or sperm viability were not affected by H2O2, although most motility parameters decreased. H2O2 decreased the percentage of chlortetracycline pattern F sperm at 4 hr and increased the percentage of acrosome-reacted sperm (pattern AR) after 1 hr. Pooled data of Experiment 2 showed LPO was positively correlated with SCSA (r = 0.29 to r = 0.59; P < 0.05 to P < 0.01), while most motility parameters and the percentage of viable sperm were negatively correlated with LPO (r = -0.30 to r = -0.38; P < 0.05 to P < 0.01). LPO was positively correlated with the percentage of pattern AR sperm (r = 0.33; P < 0.01). Cryopreservation and H2O2 promote DNA instability in ram sperm, though motility is a more sensitive indicator of oxidative stress than the other parameters investigated.     

Nuclear translocation of dihydrofolate reductase is not a pre-requisite for DNA damage induced apoptosis

Dihydrofolate reductase (DHFR) is a key enzyme for the synthesis of thymidylate, and therefore, of DNA. By applying subcellular proteomic analysis, we identified that the DHFR protein was translocated from cytoplasm into the nucleus when apoptosis was induced by NSC606985, a camptothecin analogue. The nuclear translocation of DHFR protein during apoptosis was independent of the cellular context, but it was more sensitive in cell death induction by DNA damaging agents such as doxorubicin, etoposide and ultraviolent radiation than endoplasmic reticulum stressors (brefeldin-A and tunicamycin) and anti-microtubule agents (paclitaxel and nocodozole). The addition of methotrexate almost completely blocked the nuclear translocation of DHFR protein. Further investigations showed that the nuclear translocation of DHFR was not a pre-requisite for DNA damage induced apoptosis. Therefore, its potential biological significance remains to be further explored. Ting-Ting Yuan and Ying Huang contribute equally to this work.     

Effect of prostatic fluid on the quality of fresh and frozen-thawed canine epididymal spermatozoa

Canine epididymal spermatozoa have a low freeze-tolerance ability compared with ejaculated spermatozoa, which could arise from the absence of prostatic fluid (PF). Therefore, the purpose of this work was to elucidate the influence of PF on the quality of canine epididymal sperm before and after freezing. Caudae epididymides were retrieved from eight dogs after routine castration. Spermatozoa were released by slicing the tissue and were extended in either Tris solution or PF before freezing. Frozen sperm samples were thawed at 70 °C for 8 seconds in a waterbath. Sperm concentration, motility using computer-assisted sperm analysis, morphology, plasma membrane, acrosome and chromatin integrity were assessed in the fresh sperm samples (after 20 minutes incubation) and at 0 and 4 hours after thawing. Progressive motility, distance straight line, distance average path, average path velocity, curvilinear velocity, straight line velocity, straightness, linearity, wobble, and beat cross frequency were significantly increased after extraction into PF. There was a higher proportion of spermatozoa with DNA damage in the PF treatment group at 4 hours after thawing than in the Tris treatment group (15.8% vs. 6.7%, P < 0.05). These results suggest that the addition of PF to canine spermatozoa activates sperm motility in fresh spermatozoa but has a negative effect on chromatin integrity after freezing–thawing.     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.     

Effect of dilution rate on feline urethral sperm motility,viability, and DNA integrity

This study was designed to investigate if the characteristics of feline urethral sperm can be affected by high dilution in an artificial medium. The semen collected by urethral catheterization from eight male cats was evaluated for sperm concentration and motility and subsequently diluted with a TRIS-based extender to the concentration of spermatozoa 10 × 10⁶/mL, 5 × 10⁶/mL, and 1 × 10⁶/mL. Immediately after the extension samples were assessed for motility, cell viability using SYBR-14 and propidium iodide, acrosome integrity using lectin from Arachis hypogaea Alexa Fluor 488 Conjugate, and propidium iodide and chromatin status by acridine orange. Compared with 10 × 10⁶/mL dilution rate, spermatozoa diluted to 1 × 10⁶ sperm/mL had a significantly lower proportion of motile (31.1% ± 19.8 and 0.7% ± 1.6, respectively, P < 0.05) and viable spermatozoa (88.3% ± 3.1 and 69.1% ± 12.8, respectively, P < 0.01). There was no dilution-related difference in the acrosome integrity (76.7% ± 11.9 vs. 75.9% ± 10.6) and chromatin status (defragmentation index, 3.3% ± 0.97 vs. 3.4% ± 1.7). These results indicate that feline urethral semen is susceptible to high dilution rate, and some sperm characteristics can be artifactually changed by semen dilution. It also suggests the potential role of seminal plasma in maintaining sperm motility and viability in high dilution rates.     

冷冻对山羊精子转染外源DNA和体外制备转基因胚胎的影响

本实验将鲜精和冻精分别与地高锌标记的线形化的pEGFP-N,质粒孵育转染,用原位杂交方法检测转染效率;PCR和Southern Blotting检测精子与外源DNA的整合效率;与成熟卵母细胞体外受精,PCR检测阳性胚胎比率,用透射电镜技术、碘化丙锭和羟化荧光素双探针技术和单细胞电泳（Single Cell Gel Electrophoresis,SCGE）技术,观察精子冷冻前后的超微结构、精子质膜完整性和精子核DNA损伤的变化,研究冷冻对山羊（Caprahircus）精子转染内化外源DNA和体外制备转基因胚胎的影响及机理。结果表明,冻精显著提高了转染外源DNA的效率（81．60％&#177;16．59％VS32．95％&#177;2．93％,t=4．873,P=0．003;41．80％&#177;6．26％vs27．89％&#177;8．64％,t=2．634,P=0．039）。PCR和Southern Blotting检测表明外源DNA已经整合到精子基因组上。用冻精与成熟卵母细胞体外受精,体外受精穿透率和卵裂率显著低于鲜精组（24．19％&#177;3．15％vs58．86％&#177;3．73％,t=7．131,P〈0．001;11．83％&#177;2．37％vs29．71&#177;3．47％,t=4．302,P〈0．001）,但体外生产的胚胎PCR阳性率比鲜精组显著提高（45．45％&#177;10．87％VS24．44％&#177;6．06％,t=1．750,P=0．013）。超微结构观察和双荧光探针检测都发现冷冻-解冻精子质膜完整性降低（8．34％&#177;4．21％VS65．67％&#177;6．46％,t=12．492,P〈0．001）,SCGE显示冷冻极显著增加了精子彗尾长度和彗星细胞比例（42．67μm&#177;4．56μmvs21．14／Lm&#177;2．36μm,t=5．644,P=0．005;60．00％&#177;4．00％vs17．37％&#177;2．57％;t=15．787,P〈0．001）。冷冻-解冻可以提高山羊精子转染外源DNA的效率,冷冻破坏精子质膜完整性,解除质膜的阻碍作用,是提高外源DNA转染效率的一个主要原因[动物学报54（6）：1089-1097,2008]。     

Effects of cooling, cryopreservation and heating on sperm proteins, nuclear DNA, and fertilization capability in mouse

In the present study, we used confocal microscopy and electrophoresis to study the effects of heating to 5 or 100 degrees C or cooling to 4 degrees C or -- 196 degrees C on the stability of sperm proteins and DNA. We used intracytoplasmic sperm injection (ICSI) to determine the fertilizing capability of treated spermatoza. It was shown that sperm cryopreservation at - 196 degrees C or cooling at 4 degrees C altered neither protein and DNA profiles nor the sperm fertilization capability, while the protein and DNA profiles of sperm heated at 100 degrees C were irreversibly degraded and inactivated. The proteins of sperm were severely damaged while the nuclear DNA still maintained its integrity when heated to 58 degrees C. Observation by laser confocal microscopy showed that after being heated to 58 degrees C and 100 degrees C, the nuclear of mouse sperm lost their ability to activate oocytes and they could not transform to male pronuclei though the membrane of some sperm could degrade and induce the formation of sperm asters in ICSI oocytes. The results indicate that the use of 58 degrees C heating only causes the degradation of sperm proteins, while the 100 degrees C heating elicits the irreversible degradation of both sperm proteins and nuclear DNA, and the damage of sperm proteins is primarily responsible for the observed decrease in sperm fertilizing capability.     

Effects of osmotic pressure on motility, plasma membrane integrity and viability in fresh and frozen-thawed buffalo spermatozoa

In the first experiment, osmotic pressure of semen and seminal plasma in a semen sample from each of the 20 mature Nili-Ravi buffalo bulls was determined. In the second experiment, effects of osmotic pressure on motility (%), plasma membrane integrity (%) and viability (%) in fresh and frozen-thawed semen samples from each of the seven mature Nili-Ravi buffalo bulls was determined. In the first experiment, seminal plasma was harvested by centrifuging semen at 400 × g for 10 min at 37°C and osmotic pressure was determined using an osmometer. In the second experiment, motility (%) was assessed in fresh and frozen-thawed (37°C for 30 s) semen samples using a phase-contrast microscope (×400). Plasma membrane integrity (%) was determined by mixing 50 μl each of fresh and frozen-thawed semen with 500 μl of solution having an osmotic pressure of 50, 100, 150, 190 or 250 mOsm/l (hypotonic treatments of fructose + sodium citrate) and incubating at 37°C for 1 h. Viability (%) of fresh and frozen-thawed spermatozoa before and after challenging them to osmotic pressure (hypotonic treatments) was assessed using supravital stain under a phase-contrast microscope (×400). In the first experiment, the mean ± s.e. osmotic pressures of the buffalo semen and seminal plasma were 268.8 ± 1.17 and 256.0 ± 1.53 mOsm/l, respectively. In the second experiment, motility (%) decreased (P < 0.05) in frozen-thawed semen samples as compared with fresh semen (60.1 ± 1.34 v. 81 ± 1.57, respectively). The plasma membrane integrity (%) and magnitude of osmotic stress in fresh and frozen-thawed semen samples was higher (P < 0.05) at 50, 100, 150 and 190 mOsm/l as compared with 250 mOsm/l. Loss of viability (%) in fresh and frozen-thawed semen samples was higher (P < 0.05) at 50 mOsm/l (59% in fresh, 70% frozen thawed) as compared with other osmotic pressures, while it was lowest at 250 mOsm/l (4.1% for fresh, 9.7% frozen thawed). In conclusion, osmotic pressure of Nili-Ravi buffalo semen and seminal plasma is determined. Furthermore, variation in osmotic pressure below 250 mOsm/l is not favorable to fresh and frozen-thawed buffalo spermatozoa.     

Effects of kinetin supplementation on the post-thaw motility,viability, and structural integrity of dog sperm

Oxidative stress is one of the major issues associated with cryopreservation because it causes a marked reduction in the post-thaw quality of semen. This study investigated the ability of kinetin to preserve the structural and functional integrity of dog sperm during cryopreservation. Pooled ejaculates were divided into 5 equal aliquots, diluted with buffer 2 supplemented with different concentrations of kinetin (0, 25, 50, 100, and 200 μM), and finally cryopreserved. The optimal concentration of kinetin was 50 μM based on the significantly improved (P < 0.05) motion characteristics and viability of post-thaw sperm samples. Moreover, kinetin-supplemented samples exhibited significantly higher (P < 0.05) sperm counts with the intact plasma membrane, normal acrosomes, mitochondria, and chromatin than control. The beneficial effects of kinetin were also reflected by the significant increase in the expression levels of anti-apoptotic (B-cell lymphoma, BCL2) and protamine-related genes (protamine 2, PRM2; protamine 3, PRM3), and decrease in the expression of pro-apoptotic (BCL2-associated X, BAX) and mitochondrial reactive oxygen species-modulating genes (ROS modulator 1, ROMO1) in kinetin-supplemented sperm samples than in control. The results demonstrated that supplementation of buffer 2 with 50 μM kinetin is ideal for reducing the magnitude of oxidative damage during semen cryoprocessing and improving the post-thaw quality of dog semen.