Sperm function during incubation with oestrus oviductal fluid differs in bulls with different fertility.

Spermatozoa undergo several modifications in the oviduct before acquiring fertilising capacity. Although spermatozoa are exposed to similar conditions in the oviduct, the speed of the response varies with the male and the state of the spermatozoa. We hypothesised that spermatozoa from bulls with different fertility may differ in their ability to respond to oviductal fluid (ODF). Frozen-thawed spermatozoa from four bulls were incubated with oestrus oviductal fluid (OODF) for 6h. Sperm kinematics, tyrosine phosphorylation, phosphorylation patterns, capacitation and acrosome reaction were analysed at hourly intervals. The amplitude of lateral head displacement (ALH) and straightness coefficient (STR) were higher (P<0.05) in bulls with higher fertility compared with those with lower fertility, at 1-4h of incubation. At 4h of incubation and onwards, spermatozoa from bulls with higher fertility showed a lower degree (P<0.05) of tyrosine phosphorylation and higher degree of capacitation and acrosome reaction. At least five tyrosine-phosphorylated sperm proteins were detected in all bulls. However, the expression of two phosphorylated sperm proteins (183 and 109 kDa) was upregulated in bulls with lower fertility. It may be concluded that cryopreserved spermatozoa from high- and low- fertile bulls differ in their ability to respond to OODF. This may help in developing tools for assessing fertility of bulls, once validated in more animals.

Comparison of DNA Fragmentation Assay in Frozen-Thawed Cat Epididymal Sperm.

DNA fragmentation of frozen-thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA(®) ), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p < 0.05) with no correlations. Only DFI values obtained from SCD revealed a significantly higher DFI in corpus compared with cauda spermatozoa (p < 0.05). The discrepancy between techniques might be due to the sensitivity of each technique, differences in severity of DNA damaged that can be detected. The difference in DFI between epididymal regions from SCD technique might indicate different maturational stages of spermatozoa, with less chromatin condensation of spermatozoa in corpus compared with cauda epididymis.

Macroscopic and microscopic evaluation of Eurasian lynx (Lynx lynx) female tubular reproductive organs in relation to ovarian structures.

Although monitoring wild animals in the field is essential for estimations of population size and development, there are pitfalls associated with field monitoring. In addition, some detailed data about reproductive physiology can be difficult to obtain in wild live animals. Studying reproductive organs from the Eurasian lynx killed at hunting or found dead could be used as a valuable addition to other field data. We evaluated reproductive organs from 39 Eurasian lynx females (Lynx lynx) killed in Sweden during the hunting seasons in 2009, 2010, and 2011. According to notes on ovarian structures, the animals were categorized as being in one of four different reproductive stages: juvenile (n = 10), follicular stage (n = 8), luteal stage (n = 11), and anestrus (n = 10). Corpora lutea were classified as fresh CL from the present season or as luteal bodies from previous cycles. Microscopic evaluations were blindly coded while the outer measurements of the vagina and uterus were taken at the time of organ retrieval. The width of the endometrium, myometrium, outer width of the uterine horns, and the diameter of the vagina differed significantly with the reproductive stage (P < 0.001) and were largest in the follicular and luteal phases. The number of endometrial glands evaluated blindly coded on a subjective scale was significantly associated with the reproductive stage (P < 0.0001) and was significantly higher in the luteal phase than that in any other reproductive stages (P < 0.05). Cornification of the vaginal epithelium was only observed in females in the follicular stage or in females with signs of a recent ovulation. In conclusion, both macroscopic and histologic measurements are useful for a correct classification of the reproductive stage when evaluating reproductive organs in the Eurasian lynx killed during the hunting season. Routine evaluation of reproductive organs has a potential to be a useful additional tool to field studies of live lynx to monitor their reproduction.

Blood plasma collected after adrenocorticotropic hormone administration during the preovulatory period in the sow negatively affects in vitro fertilization by disturbing spermatozoa function.

Successful fertilization is essential for reproduction and might be negatively affected by stressful events, which could alter the environment where fertilization occurs. The aim of the study was to determine whether an altered hormonal profile in blood plasma caused by adrenocorticotropic hormone (ACTH) administration could affect in vitro fertilization in the pig model. In experiment 1, gametes were exposed for 24 hours to plasma from ACTH-treated, non-ACTH-treated sows, or medium with BSA. Fertilization, cleavage, and blastocyst rates were lower in the ACTH group compared with the no ACTH or BSA control groups (P < 0.01). In experiment 2, the exposure of matured oocytes for 1 hour before fertilization to the same treatments did not have an impact on their ability to undergo fertilization or on embryo development. In experiment 3, spermatozoa were incubated for 0, 1, 4, and 24 hours under the same conditions. There was no effect of treatment on sperm viability. The percentage of acrosome-reacted spermatozoa remained higher in the ACTH group compared with the non-ACTH-treated group through the incubation period (P < 0.001). Protein tyrosine phosphorylation (PTP) patterns were also affected by treatment (P < 0.001). The presence of an atypical PTP pattern was higher in the ACTH group at all the analyzed time points compared with the BSA and no ACTH groups (P < 0.001). In conclusion, this altered environment may not affect oocyte competence but might affect the sperm fertilizing ability through alterations in the acrosome reaction and correct sequence of PTP patterns.

Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca²âº concentration in boar spermatozoa during cryopreservation.

Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.

The effect of oviductal fluid on protein tyrosine phosphorylation in cryopreserved boar spermatozoa differs with the freezing method.

Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO(2). Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study demonstrates that preovulatory isthmic ODF induce tyrosine phosphorylation in a higher proportion of boar spermatozoa compared to the post-ovulatory fluid and that the method of freezing significantly influences the response of post-thaw spermatozoa to porcine ODF.

Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate.

Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca(2+)]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P<0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca(2+)]i between P1 and SRF-P1 in fresh as well as in frozen-thawed semen. A higher (P<0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.

The effects of antioxidants on semen traits and in vitro fertilizing ability of sperm from the flat-headed cat (Prionailurus planiceps).

Since antioxidants can overcome the negative effects of reactive oxygen species (ROS) during sperm cryopreservation, post-thaw sperm quality in flat-headed cats (Prionailurus planiceps), an endangered species, might benefit from the addition of antioxidants to semen extender. The objectives of this study were to: 1) investigate semen traits; and 2) evaluate effects of the vitamin E analogue Trolox (vitamin E) and glutathione peroxidase (GPx) on the quality of frozen sperm from captive flat-headed cats in Thailand. Eight ejaculates were collected by electroejaculation from four flat-headed cats. Each semen sample was divided into three aliquots and re-suspended in a semen extender as follows: 1) without antioxidant supplementation (control); 2) supplemented with 5 mM vitamin E; or 3) supplemented with 10 U/mL GPx. All samples were cryopreserved and thawed. Subjective sperm motility, progressive motility, and the integrity of the sperm membrane, acrosome and DNA were evaluated at semen collection, after 1 h cold storage, and at 0 and 6 h after thawing. Mitochondrial membrane potential, early apoptotic cells, and embryo development by heterologous in vitro fertilization were evaluated after thawing. Captive flat-headed cats were affected by teratozoospermia. After 1 h cold storage, sperm membrane integrity in samples supplemented with GPx was higher than the control group (54.5 ± 13.7 vs 51.3 ± 13.9; P < 0.05; mean ± SD). Sperm frozen in extender with GPx had higher motility at 6 h and greater mitochondrial membrane potential at 0 and 6 h post-thaw incubation than the other groups (P < 0.05). In conclusion, GPx improved the quality of frozen-thawed sperm in flat-headed cats.

Single layer centrifugation of stallion spermatozoa through Androcoll™-E does not adversely affect their capacitation-like status, as measured by CTC staining.

This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation-like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6°C. Sperm capacitation-like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation-like status between colloid-selected and non-selected spermatozoa. Sperm motility decreased significantly during cold storage, whereas the proportion of apparently capacitated spermatozoa increased. There was no change in the proportion of acrosome-reacted spermatozoa. In conclusion, SLC through Androcoll™-E does not adversely affect the capacitation-like status of stallion spermatozoa, although it did increase with time during cold storage.

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction.

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe(2+)) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n=13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe(2+), EE-CAT plus Fe(2+), EE-GPx plus Fe(2+) and EE-SOD plus Fe(2+)). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2h after thawing (P<0.05). Catalase supplementation, however, improved DNA integrity at 4h (P<0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6h, linear motility at 6h, mitochondrial activity at 6h, membrane integrity at 2 and 6h, and DNA integrity at 4h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2h after thawing (P<0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe(2+) negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P<0.05). After thawing, there were, however, no significant differences between the control plus Fe(2+) and the antioxidative enzymes supplementation plus Fe(2+) groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.

Heparin and dermatan sulphate induced capacitation of frozen-thawed bull spermatozoa measured by merocyanine-540.

Glycosaminoglycans (GAGs) are present in the oviduct in which the major part of sperm capacitation occurs. In this study we have tested how capacitation of frozen-thawed bull spermatozoa is effected by exposure to different GAGs detectable or possibly present in oviductal fluid; i.e. heparin, hyaluronan, heparan sulphate, dermatan sulphate and chondroitin sulphate. Following exposure of different duration, the spermatozoa were stained with either Chlortetracycline (CTC) or merocyanine-540 and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Heparin elicited a significant increase in the number of alive, capacitated spermatozoa, either expressed as higher merocyanine-540 fluorescence (p < 0.0001) or as B-pattern (p = 0.0021) in the CTC assay, during 4 h of incubation. When comparing the different GAG treatments one by one to the negative control in the flow cytometric study, only heparin and dermatan sulphate were significant (p < 0.0001) higher than the control at 0-30 min of incubation. Duration of incubation did not affect the proportion of capacitated spermatozoa when measured as merocyanine-540 fluorescence or CTC B-pattern, but the length of the incubation did affect the number of dead (Yo-PRO 1 positive) spermatozoa (p < 0.0001). Exposure to zona pellucida proteins significantly increased the proportion of acrosome reacted spermatozoa (p = 0.016). Both heparin and dermatan sulphate induce capacitation of frozen-thawed bull spermatozoa in vitro.

Two different schemes of twice-weekly ovum pick-up in dairy heifers: effect on oocyte recovery and ovarian function.

The aim of the present study was to compare two different schemes of twice-weekly ovum pick-up (OPU), continuous (C) and discontinuous (DC), with special emphasis on differences in oocyte yield and quality, estrous cyclicity, ovarian dynamics, and progesterone production. Subsequent to characterization of their normal estrous cycles (pre-OPU period), eight dairy heifers were subjected to 4 months of twice-weekly OPU under two different schemes: the DC (OPU restricted to Days 0-12 of the cycle) and the C schemes. Effects of the two different schemes on oocyte yield, quality, and in vitro competence, together with effects on ovarian dynamics and progesterone production, were monitored. The mean numbers of punctured follicles and recovered oocytes per session were slightly higher (not significant (n.s.)) using the DC scheme, but in total, similar numbers of oocytes were obtained. The quality of the oocytes as well as cleavage rate after in vitro fertilization of the oocytes did not differ between the two OPU schemes. There was no influence of a corpus luteum (CL) producing progesterone on the oocyte yield and quality, whereas the presence of dominant follicles appeared to decrease the number of recovered ooctyes. During the pre-OPU period, all heifers showed normal cyclicity. In the DC scheme, the heifers showed regular and normal cyclic activity throughout the puncture period, with one to two complete follicular waves during the interval from the last OPU to the next estrus. In the C scheme, the heifers occasionally revealed cyclicities with irregular interestrous intervals and weaker signs of estrus. No complete follicular waves were seen during the OPU period in this scheme. The CL developed from the ovulation of the preovulatory follicles in the DC scheme showed similar characteristics to the CLs of the pre-OPU period; however, the CL-like structures from the puncture of follicles, in both the DC and the C schemes, revealed a shorter life span and inferior competence in producing progesterone (P<0.05). The present results indicate that the DC OPU scheme, which allows animals to go into natural ovulation prior to the first OPU, does not affect their ovarian function, whereas the C OPU scheme does. Our study further demonstrates that an equal number of oocytes can be obtained with both schemes, but that fewer OPUs are needed when the DC scheme is applied.

The frequency of micronuclei in lung cancer cell lines and their correlation to intrinsic radiation sensitivity.

BACKGROUND: Micronuclei arise from chromosomal, acentric, fragments or whole chromosomes that are not incorporated into the daughter nuclei at mitosis. The micronuclei assay is technically simple and requires only one mitosis in order to obtain information concerning the amount of micronuclei. This study was performed to investigate whether the formation of micronuclei could be used as a marker for intrinsic radiation sensitivity in lung cancer patients. MATERIALS AND METHODS: Two human lung cancer cell lines, a non-small cell lung cancer (NSCLC) cell line (U-1810) and a small cell lung cancer (SCLC) cell line (U-1906L), were used. Radiosensitivity data on the survival fraction at 2 Gy were obtained from the clonogenic assay. Radiation was delivered as one-fraction doses: 0, 1, 2, 4, 10 and 20 Gy. After irradiation, the cells were incubated for 0, 24, 48, 60, 72 and 96 hours before fixation and staining. RESULTS: The frequency of micronuclei in U-1906L was clearly elevated after 96 hours in the 20 Gy fraction. The frequency of micronuclei reached 5.5%. For U-1810 the micronuclei had a peak clearly different than the other settings after 48 hours in the 10 Gy fraction. The frequency of micronuclei was 1.2%. CONCLUSION: Counting micronuclei is not sensitive enough for estimation of radiosensitivity in clinical doses. However, our results demonstrated a distinct difference between NSCLC and SCLC cell lines at higher doses. This difference might be due to different repair fidelity, so future studies with this assay should aim to investigate this hypothesis.

Serum p53 autoantibodies as prognostic marker in patients with oesophageal carcinoma.

BACKGROUND: Oesophageal carcinoma is one of the more aggressive cancers and the patients usually seek medical attention only when the disease is already advanced. Therefore it is important to have a tool, which is simple and fast, to measure and to predict the prognosis for these patients. Mutations in the p53 gene are among the most common genetic abnormalities in oesophageal carcinoma. The present study is the first study, to our knowledge, in which the relationship between the presence of p53 autoantibodies in the serum and survival has been investigated in patients with oesophageal carcinoma. PATIENTS AND METHODS: Serum from patients with oesophagal carcinoma was collected between 1996 and 1999 at the Department of Oncology, Uppsala University Hospital, Sweden. The serum samples were analysed for the presence of p53 autoantibodies using a sandwich ELISA. RESULTS: In a multivariate analysis, the presence of p53 autoantibodies was associated with decreased survival (p=0.047). Patients with extensive disease had a poor prognosis and time to death was decreased in these patients (p=0.000022). The one-year survival was 0% for these patients if they had p53 autoantibodies compared to 36% for patients with no p53 autoantibodies and extensive disease. CONCLUSION: We conclude that the presence of serum p53 autoantibodies is associated with decreased survival for patients with oesophageal carcinoma.