Impact of seasonality,storage of semen,and sperm head‐shape on whole tissue methylation and expression of methylation responsive candidate genes in swine placenta and fetal livers from summer and winter breedings

Epigenetics includes the study of external factors that can influence the expression of genes by altering the accessibility of DNA through methylation. To investigate the epigenetic influence of season, sperm head shape, and semen storage on placental and fetal tissues, pregnancies were generated in the summer or winter using boar semen from either least or most sperm head shape change, collected during cool or warm seasons, and stored as cooled‐extended or cryopreserved. The lowest (p < 0.05) ratios of 5‐methylcytosine to 5‐hydroxymethylcytosine activity (5mC:5hmC) in fetal liver were from summer breedings and in placental tissues from winter breedings. The relative expression of placental CDH1 tended ( p < 0.10) to be greater in placenta generated from cryopreserved semen or semen collected during cool periods. The relative expression of placental GNAS was affected ( p < 0.05) by the interaction of breeding and semen collection seasons. Cryopreserved semen increased ( p < 0.05) the placental relative expression of GNAS. Placental MEST and RHOBTB3 tended ( p < 0.10) to have a greater relative expression from pregnancies generated using semen collected during cool periods used during winter breedings. Within fetal liver, the relative expression of GNAS and HGF was greater ( p < 0.05) from winter breedings. Interaction of winter breedings and least sperm head shape change tended ( p < 0.10) to have the greatest fetal liver expression of CDH1. Seasonality of semen collection, breeding, and the effect on sperm head shape change had an influence on the expression of genes with known differentially methylated regions or response to methylation activity from embryonic and extraembryonic tissues.     

Successful cryopreservation of honey bee drone spermatozoa with royal jelly supplemented extenders

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thawing quality of drone sperm. Semen samples were collected from sexually mature drones. Pooled semen was diluted with extender without RJ (control) or supplemented with different concentrations of RJ (1, 2, 4 or 8%). Sperm motility, plasma membrane functional integrity, and acrosomal integrity were evaluated. At post thaw, the highest sperm motility and acrosomal integrity rates were obtained in the RJ1 group. Functional integrity of sperm membrane was better preserved in the RJ1 and RJ2 groups compare to the other groups. The study shows that RJ supplemented extenders have beneficial effects on drone semen parameters. The results of the present study demonstrated advantage of using 1% RJ supplemented extender.     

Genotyping from semen of wild Japanese macaques (Macaca fuscata)

The noninvasive collection of animal cells is crucial for DNA analyses in wild populations that cannot be disturbed by capture. We describe the collection of 68 semen samples following copulation and masturbation events in wild habituated and nonhabituated troops of Japanese macaques on the protected island of Yakushima. We used this DNA to amplify 390 base pairs (bp) of the mitochondrial DNA control region in 16 individuals from eight troops, and found a monomorphic pattern in agreement with the low variability imposed by geographic isolation and female philopatry. We also amplified two microsatellite loci from samples collected after the resident males of a focal troop had copulated with different females. We found several different allele combinations in samples collected after the observed mating of a single male, indicating the presence of contaminant DNA, presumably from males that had previously mated with the same female. This discovery made it impossible to assign a given sample to a specific male except when the samples were recovered after masturbation events. Thus, it was not possible to test for kinship or estimate allele frequencies from the semen samples. The mixing of semen, and the pattern of sample collection observed in morphologically identified individuals support the notion that strong mating and sperm competition exists among resident and nonresident males.     

Improved Human Sperm Recovery Using Superoxide Dismutase and Catalase Supplementation in Semen Cryopreservation Procedure

The aim of this work was to evaluate the effects of ROS scavenger supplementation in human semen samples undergoing cryopreservation procedures.After screening out andrological pathologies, we selected 25 male partners of infertile couples with the following semen profile: volume >/= 2.0 ml, normal viscosity, sperm count >/=20 x 10(6)/ml, straight progressive motility (classes 1 and 2) >/= 40% (Mazzilli, Rossi, Delfino and Nofroni (1999) Andrologia 31: 187-194), atypical forms 相似文献   

Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production

The objective was to evaluate the structural and functional quality of bull sperm after sexing by flow cytometry. Frozen non-sexed (NS), sexed for X (SX) and sexed for Y (SY) sperm from four bulls was used. Frozen-thawed sperm was analyzed for motility, sperm head agglutination, morphology, capacitation, and integrity of the plasma membrane, acrosome, and chromatin. After Percoll centrifugation (45:60% gradients), the pellet was used for sperm analysis or IVF. Data were analyzed using generalized linear models (P < 0.05) and were reported as least squares means ± standard error (SEM). Based on sperm evaluations, NS sperm had better (P < 0.05) quality than sexed sperm, including higher motility and greater percentages of cells with an intact membrane and acrosome (58.0 ± 3.0, 58.2 ± 3.0, and 60.9 ± 3.3) than SX (29.6 ± 1.3, 36.0 ± 2.9, and 37.1 ± 3.3), and SY (26.2 ± 2.1, 36.4 ± 2.9, and 37.5 ± 3.3). There were no differences (P > 0.05) among groups for fertilization and cleavage rates. Similarly, blastocyst rate on Day 8 (Day 0 = day of insemination) did not differ among groups (22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9 for NS, SX, and SY, respectively). Regarding embryo development kinetics, all groups had similar developmental stages from Days 6 to 9. Although the sex-sorting procedure affected sperm characteristics, it did not significantly affect fertilization or embryo development.     

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.     

Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas)

Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean ± SD) 73.0 ± 12.2, 38.4 ± 8.8, 88.0 ± 0.1, and 59.3 ± 15.7%, respectively. A series of GnRH injections (3 x 250 μg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 ± 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 ± 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 ± 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 ± 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 ± 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species.