Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

Relationship between thirty post-thaw spermatozoal characteristics and the field fertility of 11 high-use Australian dairy AI sires

This study determined the relationship between two measures of field fertility of I I high-use Australian artificial insemination (AI) dairy bulls and thirty standard laboratory assessments of spermatozoal post-thaw viability. The two measures of field fertility used, conception rates (cCR) and non-return rates (cNRR), were both corrected for all major non-bull variables. Sperm viability assessments were conducted on semen collected within the same season as that used to derive the field fertility estimates. These assessments measured sperm concentration, motility, morphology and membrane integrity at thawing, after 2 h incubation and after the swim-up sperm selection procedure. Derivations of these measures and in vitro embryo fertilizing and developmental capacity were also determined. The Genstat Statistical Package [Genstat 5 Release 4.2 Reference Manual, VSN International, Oxford, 20001 was used to conduct an analysis of variance on the viability parameters across semen straws and bulls, and to calculate the strength of correlation between each semen parameter, cNRR and cCR in a correlation matrix. Step forward multiple regression identified the combination of semen parameters that were most highly correlated with cCR and with cNRR. The sperm parameters identified as being most predictive of cCR were the percentage of morphologically normal sperm immediately post-thaw (zeroNorm), the number of morphologically normal sperm after the swim-up procedure (nSuNorm), and the rate of zygote cleavage in vitro (Clv); the predictive equation formed by these parameters accounted for 70% of variance. The predictive equation produced for cNRR contained the variables zeroNorm, the proportion of membrane intact sperm after 2 h incubation at 37 degreesC (twoMem) and Clv and accounted for 76.5% of the variation. ZeroNorm was found to be consistent across straws and semen batches within-bull and the sperm parameter with the strongest individual predictive capacity for both cCR (P = 0.1) and cNRR (P = 0.001). Post-thaw sperm parameters can be used to predict field fertility of Australian dairy sires; the calculated predictive equations are particularly useful for identifying and monitoring bulls of very high and very low potential fertility within a group. (C) 2003 Elsevier B.V. All rights reserved.

Selection criteria in the search for a sperm donor: Behavioural traits versus physical appearance

Despite extensive literature on female mate choice, empirical evidence on women’s mating preferences in the search for a sperm donor is scarce, even though this search, by isolating a male’s genetic impact on offspring from other factors like paternal investment, offers a naturally ”controlled” research setting. In this paper, we work to fill this void by examining the rapidly growing online sperm donor market, which is raising new challenges by offering women novel ways to seek out donor sperm. We not only identify individual factors that influence women’s mating preferences but find strong support for the proposition that behavioural traits (inner values) are more important in these choices than physical appearance (exterior values). We also report evidence that physical factors matter more than resources or other external cues of material success, perhaps because the relevance of good character in donor selection is part of a female psychological adaptation throughout evolutionary history. The lack of evidence on a preference for material resources, on the other hand, may indicate the ability of peer socialization and better access to resources to rapidly shape the female decision process. Overall, the paper makes useful contributions to both the literature on human behaviour and that on decision-making in extreme and highly important situations.

Selection of X chromosome of buffaloes sperm with Percoll gradients

The aim of the present study was to evaluate the selection of X chromosome of buffaloes sperm with Percoll gradients. The stock solution of Percoll was prepared in the proportion of 1:11 (1 part of Percoll:11 parts of a solution containing KCl 1M, NaH(2)PO(4) 0.1M, NaCl 1.5M and sodium HEPES 23.8 g/ml). In order to prepare 9 different gradients were added to the stocked Percoll the A solution (glicine-yolk extender) in the following proportions: 90, 80, 72, 65, 57, 49, 34 and 25%. A sample of 0.7 ml of the fresh semen was deposited at 2 ml of Percoll 80% for the sperm wash. The precipitate was put in tube with 0.7 ml of each gradient. Then, the precipitated was washed in TES solution by centrifugation (500xg for 10 minutes), and collected again and diluted in TES solution to be freeze. The presence of the F body in the spermatozoa was observed in 58.7 +/- 5.4% of the control group and in 41.2 +/- 5.4% of the treated group (p<0.01). This result showed an increment of 17.55 of male sperm in the Percoll's group. The reduction of the centrifugation force did not improve the percentage of X sperm.

Sexual selection in Pomatoschistus – nests, sperm competition, and paternal care

Sexual selection arises through variation in reproductive success. This thesis investigates different aspects important in sexual selection, namely nest building, sperm competition, paternity and paternal care, and their mutual interrelationships. In the studied species, the sand goby (Pomatoschistus minutus) and the common goby (Pomatoschistus microps), sperm competition did arise when small males, so called sneakers, sneaked into other males nests and released sperm. They seemed to use female behaviour as their prime cue for a sneaking opportunity. However, also nest-holders, both with and without eggs, were found to fertilize eggs in the nests of other males. Clearly, nest-holding males tried to prevent other males from spreading their sperm in their nests, since they showed aggression towards such males. A nest building experiment indicated that the small nest-openings found in the sneaker male treatment were sexually selected through protection against sneaking or by female choice. Yet, no behavioural or genetical support for the hypothesis that the nest functions as a physical or visual defence, or that sneaker males prefer to sneak upon nests with wide nest-openings, were found in the other studies. Still, individual nest-holding males showed a higher mucus preparation effort inside the nest in the presence of a sneaker male than when alone. In close relatives, such mucus contains sperm, suggesting an importance in sperm competition. However, the mucus may also have pheromone and anti-bacterial functions and may constitute a mating effort, as found in other gobies. Both a behavioural and a mate choice experiment suggested that the males were not less eager to spawn in the presence of a sneaker male. Sneak intrusion did not affect nest defence, fanning or filial cannibalism, nor had paternity an effect on filial cannibalism. This and various life history aspects, together with the fact that the parasitic male only fertilized a fraction of the clutches, would predict females to ignore sneaker males. This was also the case, as the presence of sneaker males was found not to affect female spawning decision. Still, several females spawned in two nests, which coincided with parasitic spawnings, suggesting a cost of disturbance for the females and thus a substantial cost to the nest-holding males in terms of lost mating success. However, females paid attention to other traits in their choice of mate since spawning was associated with sand volume of the nest, but not with nest-opening width. Also, female (but not male) courtship was correlated with partial clutch filial cannibalism, indicating that females are able to anticipate future male cannibalism. In a partial correlation of nest opening, sand volume, male courtship display, displacement fanning and male size, a large number of traits were correlated both positively and negatively with regard to how we may expect them to be appreciated by females. For instance, males which fan well also build large nests or display intensely (but not both). Together with all the other results of this thesis, this shows the entangled selection pressures working on breeding animals, as well as the different male and female tactics employed to maximize their reproduction.

Multi-trait assessment of early-in-life female, male and genomic measures for use in genetic selection to improve female reproductive performance of Brahman cattle

Early-in-life female and male measures with potential to be practical genetic indicators were chosen from earlier analyses and examined together with genomic measures for multi-trait use to improve female reproduction of Brahman cattle. Combinations of measures were evaluated on the genetic gains expected from selection of sires and dams for each of age at puberty (AGECL, i.e. first observation of a corpus luteum), lactation anoestrous interval in 3-year-old cows (LAI), and lifetime annual weaning rate (LAWR, i.e. the weaning rate of cows based on the number of annual matings they experienced over six possible matings). Selection was on an index of comparable records for each combination. Selection intensities were less than theoretically possible but assumed a concerted selection effort was able to be made across the Brahman breed. The results suggested that substantial genetic gains could be possible but need to be confirmed in other data. The estimated increase in LAWR in 10 years, for combinations without or with genomic measures, ranged from 8 to 12 calves weaned per 100 cows from selection of sires, and from 12 to 15 calves weaned per 100 cows from selection of sires and dams. Corresponding reductions in LAI were 60-103 days or 94-136 days, and those for AGECL were 95-125 or 141-176 days, respectively. Coat score (a measure of the sleekness or wooliness of the coat) and hip height in females, and preputial eversion and liveweight in males, were measures that may warrant wider recording for Brahman female reproduction genetic evaluation. Pregnancy-test outcomes from Matings 1 and 2 also should be recorded. Percentage normal sperm may be important to record for reducing LAI and scrotal size and serum insulin-like growth factor-I concentration in heifers at 18 months for reducing AGECL. Use of a genomic estimated breeding value (EBV) in combination with other measures added to genetic gains, especially at genomic EBV accuracies of 40%. Accuracies of genomic EBVs needed to approach 60% for the genomic EBV to be the most important contributor to gains in the combinations of measures studied.

Pre- and post-copulatory sexual selection in the least killifish, Heterandria formosa

It has been only recently realized that sexual selection does not end at copulation but that post-copulatory processes are often important in determining the fitness of individuals. In this thesis, I experimentally studied both pre- and post-copulatory sexual selection in the least killifish, Heterandria formosa. I found that this species suffers from severe inbreeding depression in male reproductive behaviour, offspring viability and offspring maturation times. Neither sex showed pre-copulatory inbreeding avoidance but when females mated with their brothers, less sperm were retrieved from their reproductive system compared to the situation when females mated with unrelated males. Whether the difference in sperm numbers is due to female or male effect could not be resolved. Based on theory, females should be more eager to avoid inbreeding than males in this species, because females invest more in their offspring than males do. Inbreeding seems to be an important part of this species biology and the severe inbreeding depression has most likely selected for the evolution of the post-copulatory inbreeding avoidance mechanism that I found. In addition, I studied the effects of polyandry on female reproductive success. When females mated with more than one male, they were more likely to get pregnant. However, I also found a cost of polyandry. The offspring of females mated to four males took longer to reach sexual maturity compared to the offspring of monandrous females. This cost may be explained by parent-offspring conflict over maternal resource allocation. In another experiment, in which within-brood relatedness was manipulated, offspring sizes decreased over time when within-brood relatedness was low. This result is partly in accordance with the kinship theory of genomic imprinting. When relatedness decreases, offspring are expected to be less co-operative and demand fewer resources from their mother, which leads to impaired development. In the last chapter of my thesis, I show that H. formosa males do not prefer large females as in other Poeciliidae species. I suggest that males view smaller females as more profitable mates because those are more likely virgin. In conclusion, I found both pre- and post-copulatory sexual selection to be important factors in determining reproductive success in H. formosa.

Determinants of online sperm donor success: How women choose

Because the worldwide demand for sperm donors is much higher than the actual supply available through fertility clinics, an informal online market has emerged for sperm donation. Very little empirical evidence exists, however, on this newly formed market and even less on the characteristics that lead to donor success. This article therefore explores the determinants of online sperm donors’ selection success, which leads to the production of offspring via informal donation. We find that donor age and income play a significant role in donor success as measured by the number of times selected, even though there is no requirement for ongoing paternal investment. Donors with less extroverted and lively personality traits who are more intellectual, shy and systematic are more successful in realizing offspring via informal donation. These results contribute to both the economic literature on human behaviour and on large-scale decision-making.

Why do females mate with multiple males ? The sexually selected sperm hypothesis

One debated issues in evolutionary biology is, why in many species females mate with multiple males. Several hypotheses have been put forward, yet the benefits of multiple mating (here defined as mating with several males) remain unclear in many cases. The sperm sexual selection (SSS) hypothesis has been developed to account for the widespread occurrence of multiple mating in females. It argues that multiple mating by females may rapidly spread, when initially a small fraction of the females mate multiply, and if there is a heritable difference among males in one or several of the four characteristics: (1) the quantity of sperm they produce; (2) the success of their sperm in reaching and fertilizing an egg; (3) their ability to displace the sperm that females stored during previous mating; and (4) their ability to prevent any other male from subsequently introducing sperm (e.g., differential efficiency of mating plugs).

Testicular traits as selection criteria for young Nellore bulls

The objective of this study was to evaluate the possible use of biometric testicular traits as selection criteria for young Nellore bulls using Bayesian inference to estimate heritability coefficients and genetic correlations. Multitrait analysis was performed including 17,211 records of scrotal circumference obtained during andrological assessment (SCAND) and 15,313 records of testicular volume and shape. In addition, 50,809 records of scrotal circumference at 18 mo (SC18), used as an anchor trait, were analyzed. The (co) variance components and breeding values were estimated by Gibbs sampling using the Gibbs2F90 program under an animal model that included contemporary groups as fixed effects, age of the animal as a linear covariate, and direct additive genetic effects as random effects. Heritabilities of 0.42, 0.43, 0.31, 0.20, 0.04, 0.16, 0.15, and 0.10 were obtained for SC18, SCAND, testicular volume, testicular shape, minor defects, major defects, total defects, and satisfactory andrological evaluation, respectively. The genetic correlations between SC18 and the other traits were 0.84 (SCAND), 0.75 (testicular shape), 0.44 (testicular volume), -0.23 (minor defects), -0.16 (major defects), -0.24 (total defects), and 0.56 (satisfactory andrological evaluation). Genetic correlations of 0.94 and 0.52 were obtained between SCAND and testicular volume and shape, respectively, and of 0.52 between testicular volume and testicular shape. In addition to favorable genetic parameter estimates, SC18 was found to be the most advantageous testicular trait due to its easy measurement before andrological assessment of the animals, even though the utilization of biometric testicular traits as selection criteria was also found to be possible. In conclusion, SC18 and biometric testicular traits can be adopted as a selection criterion to improve the fertility of young Nellore bulls.

Motile sperm organelle morphology examination is stricter than Tygerberg criteria

The present study aimed to evaluate the correlation between the motile sperm organelle morphology examination (MSOME) and a well-known sperm morphology classification (Tygerberg criteria). For MSOME, spermatozoa were analysed at x8400 magnification by inverted microscope equipped with Nomarski differential interference contrast optics, Uplan Apo x 100 oil/1.35 objective lens and variable zoom lens. By Tygerberg criteria, the semen underwent morphological evaluation as described in the literature. Regression analysis demonstrated significant positive correlation between percentage of normal sperm forms by Tygerberg criteria and by MSOME (r = 0.83, P < 0.0001). However, the incidence of normal spermatozoa by Tygerberg criteria (9.4%) was significantly higher (P < 0.0001) than under MSOME (3.3%). Despite the highly positive correlation, MSOME is a much stricter criterion of sperm morphology classification, since it identifies vacuoles and chromatin abnormalities that are not evaluated with the same precision by the analysis of Tygerberg criteria. MSOME should be included among the routine criteria for semen analysis. In addition, MSOME should be used for selection of spermatozoa for intracytoplasmic sperm injection based on the already published literature, as this is a good selection tool.

Motile sperm organelle morphology examination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles

Background: Although the motile sperm organelle morphology examination (MSOME) was developed only as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in evaluation of semen quality, with potential clinical repercussions. The present study aimed to evaluate individual variations in the motile sperm organelle morphology examination (MSOME) analysis after a time interval.Methods: Two semen samples were obtained from 240 men from an unselected group of couples undergoing infertility investigation and treatment. Mean time interval between the two semen evaluations was 119 +/- 102 days. No clinical or surgical treatment was realized between the two observations. Spermatozoa were analyzed at greater than or equal to 8400 x magnification by inverted microscope equipped with DIC/Nomarski differential interference contrast optics. At least 200 motile spermatozoa per semen sample were evaluated and percentages of normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV/one or more vacuoles occupying >50% of the sperm nuclear area) were determined. A spermatozoon was classified as morphologically normal when it exhibited a normal nucleus (smooth, symmetric and oval nucleus, width 3.28 +/- 0.20 mu m, length 4.75 +/- 0.20 mu m/absence of vacuoles occupying >4% of nuclear area) as well as acrosome, post-acrosomal lamina, neck and tail, besides not presenting cytoplasm around the head. One examiner, blinded to subject identity, performed the entire study.Results: Mean percentages of morphologically normal and LNV spermatozoa were identical in the two MSOME analyses (1.6 +/- 2.2% vs. 1.6 +/- 2.1% P = 0.83 and 25.2 +/- 19.2% vs. 26.1 +/- 19.0% P = 0.31, respectively). Regression analysis between the two samples revealed significant positive correlation for morphologically normal and for LNV spermatozoa (r = 0.57 95% CI: 0.47-0.65 P < 0.0001 and r = 0.50 95% CI: 0.38-0.58 P < 0.0001, respectively).Conclusions: The significant positive correlation and absence of differences between two sperm samples evaluated after a time interval with respect to normal morphology and LNV spermatozoa indicated that MSOME seems reliable (at least for these two specific sperm forms) for analyzing semen. The present result supports the future use of MSOME as a routine method for semen analysis.

Efficacy of the motile sperm organelle morphology examination (MSOME) in predicting pregnancy after intrauterine insemination

Background: Although the motile sperm organelle morphology examination (MSOME) was developed merely as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in the evaluation of semen quality. The aim of this study was to determine the prognostic value of normal sperm morphology using MSOME with regard to clinical pregnancy (CP) after intrauterine insemination (IUI).Methods: A total of 156 IUI cycles that were performed in 111 couples were prospectively analysed. Each subject received 75 IU of recombinant FSH every second day from the third day of the cycle. Beginning on the 10th day of the cycle, follicular development was monitored by vaginal ultrasound. When one or two follicles measuring at least 17 mm were observed, recombinant hCG was administered, and IUI was performed 12-14 h and 36-40 h after hCG treatment. Prior to the IUI procedure, sperm samples were analysed by MSOME at 8400x magnification using an inverted microscope that was equipped with DIC/Nomarski differential interference contrast optics. A minimum of 200 motile spermatozoa per semen sample were evaluated, and the percentage of normal spermatozoa in each sample was determined.Results: Pregnancy occurred in 34 IUI cycles (CP rate per cycle: 21.8%, per patient: 30.6%). Based on the MSOME criteria, a significantly higher percentage of normal spermatozoa was found in the group of men in which the IUI cycles resulted in pregnancy (2.6+/-3.1%) compared to the group that did not achieve pregnancy (1.2+/-1.7%; P = 0.019). Logistic regression showed that the percentage of normal cells in the MSOME was a determining factor for the likelihood of clinical pregnancy (OR: 1.28; 95% CI: 1.08 to 1.51; P = 0.003). The ROC curve revealed an area under the curve of 0.63 and an optimum cut-off point of 2% of normal sperm morphology. At this cut-off threshold, using the percentage of normal sperm morphology by MSOME to predict pregnancy was 50% sensitive with a 40% positive predictive value and 79% specificity with an 85% negative predictive value. The efficacy of using the percentage of normal sperm morphology by MSOME in predicting pregnancy was 65%.Conclusions: The present findings support the use of high-magnification microscopy both for selecting spermatozoa and as a routine method for analysing semen before performing IUI.

What's the signification of nuclear vacuoles in human sperm?

The aim of this study was to determine the extent of DNA fragmentation and the presence of single/denatured or double stranded of DNA in sperm with large nuclear vacuoles (LNV) selected by high-magnification. A total of 30 patients had fresh semen samples prepared by discontinuous concentration gradient. Sperm with normal nucleus (NN) and LNV were selected at 8400x magnification and placed in different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double stranded DNA was identified by acridine orange fluorescence method. The percentage of DNA fragmentation in LNV sperm (29%) was significantly higher (P<0.001) than NN sperm (15.8%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono and oligonucleosomes), and single strand breaks (nicks) in high molecular weight DNA occur more frequently in LNV. Identically, the percentage denatured stranded DNA in sperm with LNV (67.9%) was significantly higher (P <0.0001) than NN sperm (33%). The high level of denatured DNA in sperm with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibers. Our results support an association between LNV sperm and DNA damage, and the routine selection and injection of morphological motile sperm at high magnification for ICSI. The adverse effect (DNA fragmentation or denaturation) leads to concern particularly about the possibility of iatrogenic transmission of genetic abnormalities. Copyright - SBRA - Sociedade Brasileira de Reprodução Assistida.