Prediction and analysis of analytical ultracentrifugation experiments for heterogeneous macromolecules and nanoparticles based on Brownian dynamics simulation.

In the prediction of sedimentation profiles in analytical ultracentrifugation, the counterflow due to diffusion must be taken into account for a proper analysis of experimental data in the determination of molecular properties. This is usually achieved by numerical solution of the Lamm equation. This paper presents an alternative approach, in which the displacement of the solute in the cell, resulting from the opposite effects of ultracentrifugal force and diffusional drift, is described by Brownian dynamics simulation of the solute particles. The formalism is developed for heterogeneous solutes, composed of several species, and implemented in computational schemes and tools. The accuracy of the procedure is verified by comparison with other methods based on the Lamm equation, and its efficiency is illustrated. The possibilities offered by the Brownian dynamics methods in the determination of solute properties and sample composition are demonstrated.

Evaluation of sperm subpopulation structure in relation to in vitro sperm-oocyte interaction of frozen-thawed semen from Holstein bulls.

The present study examined the relationship between the relative amount of high motile sperm and sperm-oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.

Motile sperm subpopulations in frozen-thawed dog semen: changes after incubation in capacitating conditions and relationship with sperm survival after osmotic stress.

In this study we investigated the changes that in vitro incubation under capacitating conditions could induce on the motile sperm subpopulations present in frozen-thawed dog semen samples. In addition, cryopreserved dog spermatozoa were exposed to CCM (canine capacitating medium) solutions of 300, 150, 100 and 75 mOsm and the proportions of live spermatozoa with swollen tails were recorded (HOST+). Finally, frozen-thawed dog semen samples were submitted to a second cycle of freezing and thawing and the overall sperm motility, as well as the motile sperm subpopulations structure, was determined. Cryopreserved dog semen samples were structured in four sperm subpopulations with different motility characteristics: Subpopulation (Sp) 1 contained moderately rapid and progressive spermatozoa (25.2 ± 8.5%), Sp 2 included poorly motile and non progressive sperm (15.3 ± 8.1%), Sp 3 was represented by moderately slow non progressive sperm (14.9 ± 5.9%), and Sp 4 contained the most rapid and progressive sperm (20.8 ± 14.7%). After 3h of incubation under capacitating conditions, percentages of spermatozoa assigned to Sp 2 (6.1 ± 3.4%) and 3 (4.9 ± 2.8%) significantly decreased, whereas those assigned to Sp 1 (17.0 ± 11.2%) and 4 (16.2 ± 12.8%) did not significantly change. Significant correlations were found between percentages of HOST+, for the 3 osmolarities tested, and percentages of spermatozoa included in Sp 1 and 4 after 3 h of incubation in capacitating conditions or in Sp 4 after double freezing and thawing. These results indicated that subpopulations with the most rapid and progressive sperm seemed to be highly resistant to in vitro incubation in capacitating conditions and to osmotic stress, suggesting they are likely to be the source of the fertilizing population.

Use of ultrasound in the reproductive management of dairy cattle.

The significant decrease in fertility observed in dairy cattle during the last few decades and increasing requirements by the farmers have made a regular control of reproduction indispensable to urgently identify and solve potential problems affecting reproductive efficiency. Traditionally, the main diagnostic methods used for reproductive control in cattle included rectal palpation, inspection of vaginal discharge and vaginoscopy. Since the 1990 s, the use of ultrasound (US) has become a common diagnostic method as a result of the new advances made in the development of US scans: smaller size, high level of autonomy, high image quality and accessible prices. Ultrasound improves accuracy in the diagnoses of stages of the oestrous cycle, ovarian and uterine pathologies, and pregnancy diagnosis. In addition, it facilitates the diagnosis of alterations during pregnancy (embryo mortality, foetal malformations, etc.) and helps determining foetal sex from day 55 of pregnancy.

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls.

The aim of this study was to identify different motile sperm subpopulations in ejaculates from an autochthonous bull breed (Bos taurus) and to determine possible modifications in these subpopulations resulting from cryopreservation. Ejaculates were collected and cryopreserved following a conventional protocol. The overall sperm motility and the kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates, after 4h at 5 degrees C, and at 0 and 2h postthaw. A multivariate clustering procedure separated 23,585 motile spermatozoa into four subpopulations: Subpopulation 1 showed medium velocity (VCL: 99.4+/-17.8 microm/sec) and high progressiveness (LIN: 65.1+/-14.0%); Subpopulation 2 included spermatozoa with high velocity (VCL: 148.7+/-25.6 microm/sec) but a nonprogressive trajectory (LIN: 33.1+/-10.5%); Subpopulation 3 represented slowly motile (VCL: 58.3+/-24.3 microm/sec) and nonprogressive sperm (LIN: 39.6+/-18.3%); and Subpopulation 4 included very rapid (VCL: 152.8+/-25.7 microm/sec) and highly progressive sperm (LIN: 70.9+/-13.7%). Subpopulation 4 was present in the greatest quantity in fresh ejaculates (36%), but after cooling, it significantly decreased (21%) concomitantly with an increase (P<0.001) in Subpopulation 2 (from 21% in fresh to 34% in postcooled semen). After freezing and thawing, the overall sperm motility was reduced, mainly due to Subpopulation 2 decreasing from 34% after cooling to 14% after thawing. Differences among bulls in the frequency distribution of spermatozoa within subpopulations were evidenced after thawing by different proportions of spermatozoa in Subpopulations 2 and 4. The current results indicate that a structure of four sperm subpopulations may be a common characteristic of bovine ejaculates and that the cooling phase of cryopreservation seems to be the determinant of postthaw semen quality.

Reproductive performance of rabbit does artificially inseminated via intravaginal administration of [des-Gly 10, D-Ala6]-LHRH ethylamide as ovulation inductor.

This study was aimed to evaluate the reproductive performance of rabbit does artificially inseminated (AI) with a GnRH analogue [des-Gly10, D-Ala6]-LHRH. ethylamide to induce ovulation by intravaginal administration, delivered in the seminal dose. In a preliminary experiment, 39 does were divided into three groups (n = 13) that, at the time of AI, received the following ovulation induction treatments: (i) control group: 20 microg of gonadorelin administered intramuscularly; (ii) 25 microg of the GnRH analogue added to the seminal dose; (iii) 30 microg of the GnRH analogue added to the seminal dose. Fertility did not differ between the three groups (control: 80.6%, group 2: 82.8%, group 3: 73.3%). In a second experiment, a large-scale field trial was conducted to test the use of 25 microg of the GnRH analogue [des-Gly10, D-Ala6]-LHRH ethylamide delivered in the seminal dose (n = 270) against 20 microg of gonadorelin administered intramuscularly. Fertility was higher (p < 0.05) when ovulation was induced by intravaginal administration of the GnRH agonist (91.1% vs 85.6%). Prolificacy or mortality at birth was never affected by the ovulation induction treatments. In a third experiment, two groups of does [control group (n = 39): ovulation was induced using 20 microg of gonadorelin administered intramuscularly; treatment group (n = 40): ovulation was induced using 25 microg of [(des-Gly10, D-Ala6)-LHRH ethylamide added to the seminal dose] were inseminated at 42-day intervals for five successive AI cycles, to test the response to the GnRH agonist after repeated intravaginal administration to the same animals. Fertility and prolificacy were not influenced by the ovulation induction treatment neither there was an interaction between treatment and parity. The last experiment was aimed to determine whether it could be possible to add the GnRH agonist to the semen in the AI Center, just after semen collection and dilution, or it would have to be added in the farm, immediately before AI. Kindling rates did not significantly differ when ovulation was induced by intramuscular injection of gonadorelin (84.5%) or when the GnRH agonist was added to the seminal dose just at the moment (93.8 %) or 24 h before AI (90.4 %), but it was significantly lower when the hormone was added to the semen 32 h before AI (76.3 %). Prolificacy, however, was not influenced by the ovulation induction treatment.

Effect of different thawing rates on post-thaw sperm viability, kinematic parameters and motile sperm subpopulations structure of bull semen.

The aim of the present study was to evaluate three thawing rates for bull semen frozen in 0.25-ml straws: placing the straws in a water bath at 37 degrees C for 40s, at 50 degrees C for 15s or at 70 degrees C for 5s. In a first experiment, the three thawing rates were compared in relation to post-thaw sperm motility, determined subjectively, and sperm plasma and acrosomal membrane integrity, examined by flow cytometry, after 0 and 5h of incubation at 37 degrees C. In a second experiment, the three thawing rates were evaluated based on post-thaw sperm motility, determined using a CASA system, after 0 and 2h of incubation at 37 degrees C. In addition, for the motile spermatozoa, the individual motility descriptors were analysed using a multivariate clustering procedure to test the presence of separate sperm subpopulations with specific motility characteristics in the thawed bull semen samples. Finally, it was investigated if the thawing rate had any influence on the relative frequency distribution of spermatozoa within the different subpopulations. In terms of overall post-thaw motility or plasma and acrosomal sperm membrane integrity there were no significant differences between the three thawing methods evaluated. The statistical analysis clustered all the motile spermatozoa into four separate subpopulations with defined patterns of movement: (1) moderately slow and progressive sperm (27%); (2) "hyperactivated-like" sperm (15.4%); (3) poorly motile non-progressive sperm (34.3%); (4) fast and progressive sperm (23.3%). The thawing rate had no significant influence on the frequency distribution of spermatozoa within the four subpopulations, but there was a significant effect (P<0.05) of the interaction between thawing rate and incubation time. Higher proportions of spermatozoa with fast and progressive movement were observed after 2h of post-thaw incubation when the thawing was at the faster rates (35 degrees C/40s: 8.3%, 50 degrees C/15s: 18.1% and 70 degrees C/5s: 16.5%). Whether this subtle difference might affect to the in vivo fertility of the thawed bovine semen is not known.

Sex ratio in rabbits following modified artificial insemination.

The possibility of modifying the sex ratio of rabbit litters was examined in two experiments involving artificial insemination (AI) with fresh semen. Three time periods of AI, relative to ovulation, were used in Experiment 1: (a) control, GnRH was administered immediately after AI with ovulation estimated to occur 10-12h after AI; (b) early AI, GnRH was given 6h after AI so that ovulation was delayed until 16-18 h after AI; (c) late AI, GnRH was administered 6h before AI, which was performed 4-6h before ovulation. There were 13 does per treatment, and each doe was used in the same treatment for three AIs at 42-day intervals. The second experiment involved two treatments in which the does were inseminated as for the control in Experiment 1 and AI was performed using semen prepared in the normal manner (Treatment 1) or after centrifugation through 11 discontinuous Percoll gradients (Treatment 2). There were 20 does per treatment, and each doe was used in the same treatment for three AIs at 42-day intervals. The proportion of female kits produced in Experiment 1 was: control 41.7+/-19.1%, early AI 49.8+/-17.8%, and late AI 41.4+/-16.4%. These proportions did not differ significantly between treatments or from the expected 50:50 sex ratio. Fertility was reduced by the early (60.0%) and late (73.7%) AI treatments relative to control AI (80.0%), and the difference between early and control AI almost achieved statistical significance (P<0.07). In Experiment 2, the proportion of female kits was not affected by treatment (control, 51.1%; Percoll, 54.8%), and there was a similar level of fertility for both treatments (control, 76.0%; Percoll, 74.1%). Prolificacy and perinatal mortality were not affected by treatment in either experiment. It was concluded that neither the timing of insemination nor Percoll centrifugation of semen affected the sex ratio at birth of rabbit litters.