Comet assay of fresh and cryopreserved bull spermatozoa

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

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

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30-60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 × 10⁶ sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 × 10⁶ sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 × 10⁶ sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.     

Effect of some permeating cryoprotectants on CASA motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility because they provide objective results for thousands of mammalian spermatozoa. Mammalian spermatozoa experience osmotic stress when the glycerol is added to the cells prior to freezing and removal from the cells after thawing. In order to minimize osmotic damage, cryoprotectants having lower molecular weights and greater membrane permeability than glycerol, were evaluated to determine their effectiveness for cryopreserving bull spermatozoa. The aim of this study was to compare the cryopreservation effects of low molecular weight cryoprotectants (ethylene glycol and methanol) to glycerol, on post-thaw CASA sperm parameters. Bull semen was diluted with tris-egg yolk extender containing 3% glycerol, 3, 2 and 1% ethylene glycol or 3, 2 and 1% methanol. Bull semen was frozen in 0.5 straws. Bull spermatozoa exhibited higher percentages (p<0.01) for total (Mot, 72.4%) and progressively (Prog, 29.5%) motilities when frozen in extender containing 3% glycerol compared to 3, 2 and 1% ethylene glycol or 3, 2 and 1% methanol. In conclusion, no advantages were found in using ethylene glycol or methanol to replace glycerol in bull semen freezing. Glycerol provided the best sperm characteristics for bull spermatozoa after freezing and thawing. The possibility of using ethylene glycol or methanol as permeating cryoprotectants for bull semen deserves further investigation, and these cryoprotectants should also be evaluated in extenders that contain disaccharides or cholesterol.     

Role of nitric oxide on quality of freshly ejaculated bull spermatozoa during heparin-induced in vitro capacitation

The objective of this study was to assess the effects of nitric oxide (NO) on heparin-induced capacitation in vitro of fresh bull sperm, through the addition of Nω-nitro-l-arginine methyl ester (L-NAME, a NO-synthesis inhibitor) and l-arginine (L-Arg, a NO-synthesis precursor) to the capacitation medium. In Experiment 1, different concentrations of L-NAME (0.1, 1, 10 mM) and of L-Arg (10 mM) were added to the capacitation medium. Sperm motility and vigor were subjectively appraised using direct light microscopy; sperm membrane integrity was examined using a 2% Trypan blue solution while the concentration of nitrate/nitrite (NO₃⁻/NO₂⁻) was determined by using the Griess method over a 5 h capacitation period. The addition of 10 mM L-NAME has inhibited NO synthesis, sperm progressive motility, sperm vigor and sperm membrane integrity (P < 0.05) as compared to control. The addition of 10 mM L-Arg to the capacitation medium increased all variables evaluated in comparison to the control (P < 0.05). In Experiment 2, mitochondrial activity was assessed through the MTT test (3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and sperm capacitation was assessed through the test of penetration in homologous oocytes after addition of the 10 mM L-NAME, and of the 10 mM L-Arg. The addition of 10 mM L-NAME caused mitochondrial activity (40%) and the percentage of oocytes penetrated (77%) to decrease in relation to the control (P < 0.05). After addition of 0.6 mM L-Arg + 10 mM L-NAME, partial reversal of mitochondrial activity did occur (only 20%). The addition of 10 mM L-Arg increased the percentage of oocytes penetrated as compared to control (21%) (P < 0.05). These results indicate that: (1) NO is involved in control of progressive sperm motility, vigor, membrane integrity, and mitochondrial activity along the period of heparin-induced capacitation of fresh bovine sperm via NOS/NO; (2) adequate L-Arg/NO concentrations into the capacitation medium can potentiate heparin action or act independently for increasing the number or the quality of capacitated sperm.     

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

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

The effect of sperm concentration in the ejaculate on morphological traits of bull spermatozoa

Experiments were performed on 75 ejaculates obtained from 19 bulls representing different cattle breeds used at the Masovian Centre for Animal Breeding and Reproduction in ?owicz. Fresh ejaculates were measured in respect to their volume and sperm count in the ejaculates was determined. The ejaculates were classified based on the criterion of sperm concentration and divided into five groups. Sperm morphometric measurements were taken from each bull and assessment of semen morphology was done on the basis of examination under a microscope using preparations made from fresh ejaculates. For each slide, morphometric measurements were taken of 15 randomly selected spermatozoa characterised by normal morphology and well visible under the microscope. Additionally, in each preparation morphometry of 500 spermatozoa was evaluated, numbers of spermatozoa with normal morphology and morphological abnormalities were recorded and these were categorized into spermatozoa with major and minor defects. An insignificant correlation was observed between the sperm concentration in the ejaculate and morphological traits, dimensions and shapes of bull spermatozoa. The less concentrated ejaculates contained spermatozoa with a slightly larger head circumference and a more elongated head shape in comparison with the spermatozoa in the more concentrated ejaculates. The highest frequency of morphologically malformed spermatozoa, both in the case of primary and secondary alterations, was observed in ejaculates with sperm concentration of no more than 1000 x 10(3)/mm3.     

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

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

Maintenance of fertility in cryopreserved Indian gerbil (Tatera indica) spermatozoa

This study is the first attempt at sperm cryopreservation, as well as a further examination of frozen sperm fertility by the hamster test, applied to the maintenance of an Indian gerbil (Tatera indica) colony, which is a newly developing experimental animal.The osmotic tolerance of the spermatozoa was initially investigated by subjection to hypertonicity, up to 620 mOsm/kg, for 5 min at room temperature prior to freezing. Although the percentage of total motile sperm was not affected, that of progressive motile spermatozoa began to drop at 400 mOsm/kg, and a significant decrease was observed at 620 mOsm/kg (p < 0.01). According to these results, the osmolality of the solutions for the freezing experiment, in which 6–22% raffinose was present, was fixed at approximately 400 mOsm/kg. Sperm, suspended in a plastic straw, were frozen in liquid nitrogen vapor for 5 min, followed by immersion in liquid nitrogen. Motile sperm were recovered from all freezing conditions, and high survival was obtained when sperm were frozen in the presence of 14% and 18% raffinose, with a normalized motility higher than 40%. Fertility of cryopreserved Indian gerbil sperm was examined by the zona-free hamster test. Thawed sperm adhered to 88% of the zona-free hamster oocyte surface, and some oocytes were penetrated and exhibited swollen sperm heads or male pronuclei, which we used to define fertilization. Although the fertilization rate of cryopreserved sperm to zona-free hamster eggs was significantly lower than that of fresh sperm (6% vs. 30%, p < 0.01), we demonstrated that thawed Indian gerbil spermatozoa have the ability to maintain their fertility.     

Rat embryo quality and production efficiency are dependent on gonadotrophin dose in superovulatory treatments

The present study was performed to optimize a superovulation protocol in rats in order to produce a large number of good-quality embryos suitable to develop rat embryonic stem (rES) cells. We first evaluated the ovulation kinetics of three rat strains: Wistar, Fisher and ACI/N. Animals (n=30 per strain) were treated with 50 IU of pregnant mare serum gonadotrophin (PMSG), and ovulation was induced with 50 IU of human chorionic gonadotrophin (hCG) 50 h apart. Next, we evaluated the dose-response curves of PMSG and hCG in Wistar rats in order to obtain the highest number of embryos. The parameters evaluated for superovulation efficiency were: percentage of mated females, percentage of pregnant females and the average number of embryos collected per female. The results of these experiments suggested that the best dose combination was 50 IU for each hormone. Subsequent experiments, again with Wistar rats, were designed to test which of four hormonal combination treatments (30/30, 30/50, 50/30, and 50/50 IU of PMSG/hCG) will produce the largest numbers of good-quality embryos. Embryo quality was evaluated by embryo development uniformity, embryo morphology, embryo survival in an in vitro culture and embryo ability to generate rES-like cells. Results from these experiments showed that 30/50 IU of PMSG/hCG was the treatment that induced the best embryo quality. In conclusion, our results indicated that, in Wistar rats, the most appropriate hormonal combination dose for superovulation protocols with high number of good-quality embryos was 30 IU of PMSG and 50 IU of hCG given 50 h apart. We are performing further studies with rES-like cells produced with the present methodology to evaluate if they are able to participate in the production of germ-line chimeras.     

Lipid peroxides, spermatozoa quality and activity of glutathione peroxidase in bull semen

In bull semen spontaneous lipid peroxidation measured by the level of endogenous lipid peroxides and the consequences of this process for morphological and biochemical changes was studied. Glutathione peroxidase activity as protective enzyme against peroxidative damage was also determined. Obtained results showed that approximately two thirds of GSH-Px activity in bull semen was non Se-dependent glutathione peroxidase activity. Malonaldehyde (MDA) level was negative correlated with selenium-dependent GSH-Px activity (r = = -0.38, P less than 0.01). Spermatozoa with acrosome entirely lost appeared to increase as the MDA level increased (r = 0.18, P less than 0.05). The negative correlation between Se GSH-Px activity and spermatozoa with acrosome separation from head (r = -0.28, P less than 0.01) and entirely lost (r = -0.21, P less than 0.05) suggest that selenium-dependent GSH-Px plays role in protecting the acrosome against disruption of the acrosomal membrane. The total glutathione peroxidase activity was unrelated to studied variables of bull semen.     

Effect of heat shock on function of frozen/thawed bull spermatozoa

Deposition of spermatozoa in the reproductive tract of hyperthermic cows could conceivably result in sperm damage. Accordingly, a series of experiments tested the effects of heat shock on functional characteristics and free radical production of bull spermatozoa. Viability was reduced slightly by short-term (1 to 3 h) culture at 42 and 43 degrees C as compared with culture at 39 degrees C. There was no effect of culture at 42 degrees C on the ability of spermatozoa to undergo swim-up or of 42 degrees C on the percentage of motile spermatozoa. However, exposure to 41 degrees C for 3 h reduced percentage of motile sperm, 41 and 42 degrees C reduced sperm velocity and 43 degrees C decreased the proportion of spermatozoa undergoing swim-up. In other experiments, there was no effect of heat shock (41 or 42 degrees C for 1 to 3 h) on DNA integrity, presence of intact acrosomes, or fertilizing ability of the spermatozoa. Superoxide production by spermatozoa was higher at 42 degrees C than at 39 or 41 degrees C, but there was no detectable hydrogen peroxide production at any temperature. The antioxidant, glutathione, tended to improve the ability of spermatozoa to undergo swim-up at 39 degrees C but not at 43 degrees C. Taken together, these results suggest that heat shock of a magnitude similar to that seen in vivo (41 to 42 degrees C) has little effect on sperm functions that affect fertilizing capability.     

Positive effect of butylated hydroxytoluene (BHT) on the quality of cryopreserved cat spermatozoa

The semen cryopreservation processes are associated with state of oxidative stress induced by high levels of reactive oxygen species (ROS), causing damage to functional spermatozoa. Whereby, antioxidants have been utilized to scavenge or neutralize the elevated levels of ROS. The aim of at the present study was to evaluate the effect of adding BHT to the freezing extenders on post-thaw characteristics of domestic cat spermatozoa. Semen samples were frozen in Tris-fructose-citric acid-based extender, supplemented with different concentrations of BHT (0.5 mM, 1.0 mM and 2.0 mM) and a control sample without antioxidant. After thawing, sperm samples were assessed for motility by computer‐assisted sperm analysis and viability, acrosome integrity, superoxide anion production and membrane lipid peroxidation status by flow cytometry. In the study, the parameters of sperm motility and acrosome integrity were significantly higher in 2.0 mM BHT compared to sperm frozen in the extender with other concentrations and control (P < 0.05), in addition, this concentration reduced significantly the superoxide anion production and lipid peroxidation of the sperm. The results demonstrated that the supplementation of BHT to the freezing extender could protect the function and cellular structure of domestic cat sperm from cryoinjuries.     

Modelling epistatic effects of embryo and endosperm QTL on seed quality traits

Coordinated expression of embryo and endosperm tissues is required for proper seed development. The coordination among these two tissues is controlled by the interaction between multiple genes expressed in the embryo and endosperm genomes. In this article, we present a statistical model for testing whether quantitative trait loci (QTL) active in different genomes, diploid embryo and triploid endosperm, epistatically affect a trait expressed on the endosperm tissue. The maximum likelihood approach, implemented with the EM algorithm, was derived to provide the maximum likelihood estimates of the locations of embryo- and endosperm-specific QTL and their main effects and epistatic effects. This model was used in a real example for rice in which two QTL, one from the embryo genome and the other from the endosperm genome, exert a significant interaction effect on gel consistency on the endosperm. Our model has successfully detected Waxy, a candidate gene in the embryo genome known to regulate one of the major steps of amylose biosynthesis in the endosperm. This model will have great implications for agricultural and evolutionary genetic research.     

Effect of insulin-like growth factor-I on some quality traits and fertility of cryopreserved ovine semen

The objective was to evaluate the effects of insulin-like growth factor-I (IGF-I) on the quality and fertility of frozen/thawed ovine semen. Five rams (five ejaculates/ram) were used for evaluation of semen parameters. Before cryopreservation, ejaculates were divided into four aliquots and extended with Tris alone or supplemented with human IGF-I (50, 100, or 250 ng/mL). Semen was evaluated immediately after thawing (T0), after 1 h (T1) and 2 h (T2) post-incubation at 37 °C. The percentage of live cells (fluorescence analysis-calcein and ethidium), acrosome integrity (NAR) and motility were analyzed, and hypo-osmotic swelling tests (HOST) were used to evaluate membrane resistance. In addition, AI was performed using 121 ewes to compare the optimal concentration of IGF-I vs. Tris alone on pregnancy rates after laparoscopic insemination. Pregnancy diagnosis was performed by transrectal ultrasonography. After 1 and 2 h post-incubation, in every group, percentage motile sperm, NAR and HOST decreased compared to semen at T0. Motility was higher (P < 0.05) in the IGF-I 100 and IGF-I 250 groups when compared to the IGF-I 50 and Tris groups (76.2 and 74.4% vs. 66.2 and 64.4 percent, respectively) at T0, after 1 h (67 and 63.6% vs. 56.2 and 54.7%) and 2 h post-incubation (58.2 and 55.8% vs. 48 and 47.2%). Furthermore, viability was higher (P < 0.05) in the insulin-like growth factor-I (IGF-I) 100 and IGF-I 250 groups than in the IGF-I 50 and Tris groups (88.7 and 88.3% vs. 76.6 and 77.6%, respectively) at T0. There was no difference (P > 0.05) in NAR or hypo-osmotic swelling tests (HOST) among groups. There were no differences (P > 0.05) in fertility between the IGF-I 100 and Tris groups. In conclusion, IGF-I improved subjective sperm motility and structural integrity of the plasma membrane without a significant effect on 45-day pregnancy rates after laparoscopic insemination of ewes with frozen-thawed semen.