Within-subject variability of human semen in regard to sperm count, volume, total number of spermatozoa and length of abstinence

The within-subject variability of the semen sperm count (n), volume (v) and the total number of spermatozoa (N) was studied on 220 ejaculates from 36 normal subjects after an abstinence of 7 days or less. For each of the three variables, the within-subject standard deviation, sigma, was practically proportional to the mean, mu; the common value of the coefficient of variation sigma/mu for all subjects was very high: 0.39 for n, 0.28 for v and 0.55 for N. The 95% confidence intervals based on a single ejaculate were asymmetrical and very large, the lower and upper limits being respectively 0.5 x n and 2.3 x n; 0.7 x v and 1.8 x v; 0.4 x N and 2.9 x N. The three semen characteristics for a given subject were highly correlated with length of abstinence: for an increase in abstinence of 1 day there were mean increments of 13 x 10(6)/ml for n, 0.4 ml for v, and 87 x 10(6) for N.     

Sex ratio determination in bovine semen: a new approach by quantitative real time PCR

Sex preselection of livestock offspring in cattle represents, nowadays, a big potential for genetic improvement and market demand satisfaction. Sperm sorting by flow cytometer provides a powerful tool for artificial insemination and production of predefined sexed embryos but, an accurate verification of the yield of sperm separation remains essential for a field application of this technique or for improvement and validation of other related semen sexing technologies. In this work a new method for the determination of the proportion of X- and Y-bearing spermatozoa in bovine semen sample was developed by real time PCR. Two sets of primers and internal TaqMan probes were designed on specific X- and Y-chromosome genes. To allow a direct quantification, a standard reference was established using two plasmid cDNA clones (ratio 1:1) for the specific gene targets. The method was validated by a series of accuracy, repeatability and reproducibility assays and by testing two sets of sorted and unsorted semen samples. A high degree of accuracy (98.9%), repeatability (CV=2.58%) and reproducibility (CV=2.57%) was shown. The results of X- and Y-sorted semen samples analysed by real time PCR and by flow cytometric reanalysis showed no significant difference (P>0.05). The evaluation of X-chromosome bearing sperms content in unsorted samples showed an average of 51.11+/-0.56% for ejaculates and 50.17+/-0.58% for the commercial semen. This new method for quantification of the sexual chromosome content in spermatozoa demonstrated to be rapid and reliable, providing a valid support to the sperm sexing technologies.     

Effect of glutaraldehyde concentration and fixative temperature on the number of spermatozoa with normal acrosomes in goat semen

Two experiments were conducted to determine the best fixative solution and the most suitable temperature for fixing sperm cells from goat ejaculates. In Experiment 1, a 6x3 factorial design was used to test 4 glutaraldehyde concentrations (0.5, 1, 2 and 4%) plus 1 treatment that did not contain a fixative (0%) but to which 0.3% sodium fluoride (NaF) had been added to immobilize the spermatozoa; the control treatment contained no fixative or NaF. The 6 treatments were tested with 3 different solvents: PBS, Na citrate and BL-1, representing a total of 18 samples per replicate. The fixed samples always provided a significantly higher (P<0.01) percentage of normal acrosomes than the unfixed samples, whether immobilized with NaF or observed inmediately after dilution. In Experiment 2, a 3x3 factorial design was used to determine the effect of the temperature of the glutaraldehyde fixative solution on the number of morphologically normal acrosomes from goat semen samples kept at 3 different temperatures. Our findings indicated that at all 3 fixative solution temperatures (5, 20 and 37 degrees C) there was a significant difference (P<0.01) in the percentage of normal acrosomes. At 5 degrees C, glutarhaldehyde yielded a general mean number of 53.6 normal acrosomes vs 75.1 at 20 degrees C and 83.05 at 37 degrees C. Based on these results, we recommend that the temperature of a fixative solution be established when designing an experiment using goat semen, since the temperature has a significant effect on the number of the normal acrosomes found in a semen sample.     

Relationship between bovine fertility and the number of spermatozoa penetrating the cervical mucus within straws

In this study, by using a recently developed test technique, the relationship between the total spermatozoa number penetrating determined sites of bovine cervical mucus in straws and potential fertility of bulls, and other spermatological characteristics were investigated. Furthermore, we aimed to determine the effect on the test results, of two different incubation temperatures (37 and 41 degrees C) and two sperm penetration distance ranges (PDRs). Frozen semen samples of six Holstein bulls were used in the study. The bulls were divided into two fertility groups (high and low fertility) according to the "non-return rates" (NRR). For the penetration test, cervical mucus was drawn into transparent plastic straws and incubated with semen at 37 and 41 degrees C for 15 min. After the incubation, straws were frozen in liquid nitrogen vapour and stored at -20 degrees C. On the evaluation day, concentrations of spermatozoa penetrated to the PDRs, each of which was 2.5 mm, between 32.5 and 35 mm (first penetration distance range, PDR1), and 50 and 52.5 mm (second penetration distance range, PDR2) distance in the straws from the open end, were measured. When compared with the low fertility group, bulls from the high fertility group showed a higher number of spermatozoa at the determined PDRs, and a significant positive correlation was found between the total number of spermatozoa at the penetration distances and the NRR scores of the bulls.     

Metabolism of fatty acids by bovine spermatozoa

The incorporation of (14)C-labelled myristic, palmitic, stearic, oleic and linoleic acids in vitro into the lipids of bovine spermatozoa was measured at intervals from 2min to 2h. All acids were rapidly incorporated into diglycerides, myristic acid being metabolized to the greatest extent. Whereas the low incorporation of acids into total phospholipids reflected the relative stability of the major phospholipid fractions in sperm, the minor phospholipids, particularly phosphatidylinositol, showed comparatively high metabolic activity. Although, in general, saturated acids were incorporated more actively than unsaturated substrates, stearic acid was poorly incorporated into all lipids except phosphatidylinositol. In regard to fatty acid composition of sperm lipids it was notable that diglycerides contained myristic acid as the major component, and this acid was also a prominent moiety of phosphatidylinositol. Docosahexaenoic acid was the principal fatty acid of the major phospholipid classes. These findings have been discussed in relation to the role of lipids in the metabolism of spermatozoa.     

Capacitation of bovine spermatozoa by oviduct fluid

Oviduct fluid collected from chronically cannulated oviducts of heifers was evaluated for its effect on capacitation of bovine sperm in vitro. Capacitation was determined by the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine (LC). After incubation of sperm with 0-25% (v/v) estrual oviduct fluid (collected +/- 1 day from estrus) for 4 h, addition of LC (100 micrograms/ml) for an additional 0.25 h resulted in an increasing percentage of acrosome-reacted sperm as the concentration of oviduct fluid increased. Sperm incubated 4 h with 25% estrual oviduct fluid fertilized more oocytes than sperm incubated in medium alone (p less than 0.05) but was not different from sperm incubated with 10 micrograms/ml heparin (p greater than 0.05). Glucose inhibited the ability of LC to induce ARs in sperm incubated 4 h with heparin or estrual oviduct fluid. Incubation of sperm with 25% oviduct fluid collected at various days over the estrous cycle demonstrated that peak capacitating activity was found at estrus but was also present +/- 1 day from estrus. The active capacitating factor in oviduct fluid was found to be heat stable. In addition, when extraction procedures were applied in sequential order, oviduct fluid capacitating activity was resistant to protease digestion, precipitable by ethanol, size-excluded by Sephadex G-25, and destroyed by nitrous acid. These results suggest that a heparin-like glycosaminoglycan from the oviduct is a potential in vivo capacitating agent in the bovine.     

Head-to-head association in bovine spermatozoa induced by catecholamines

Noradrenaline, adrenaline, and isoproterenol induce head-to-head association in bovine spermatozoa in a Tris-HCl-buffered medium. Noradrenaline was the most and isoproterenol the least efficient. This effect was stimulated by Ca2+, Mg2+ and Mn2+, Ca2+ being more efficient than the other two ions. At 2 X 10(-6) M Ca2+, oxidation products of adrenaline dissociated spermatozoa associated by washing; at 2 X 10(-5) M Ca2+, the dissociating effect was transformed into association. The induction of association by adrenaline was blocked by both alpha- and beta-adrenergic blockers at low concentrations (2 X 10(-7) M). Both cAMP and dibutyryl substituted cAMP (db-cAMP) induced association in the Tris-buffered medium at 2 X 10(-6) M Ca2+. Further increase in association was brought about by increasing the Ca2+ concentration to 2 X 10(-5) M. Prolongation of the treatment with cAMP increased association. When combined with cAMP under the same conditions as used in the combination with adrenaline, L-propranolol did not inhibit association induced by cAMP. In an identical experiment, performed in Tyrode solution, L-propranolol inhibited association induced by cAMP. At 2 X 10(-5) M, theophylline, caffeine, and papaverine induced association in the presence of 2 X 10(-5) M Ca2+. The results are compatible with the hypothesis that catecholamines act via receptors and formation of cAMP.     

Capacitation of bovine spermatozoa by lysophospholipids and trypsin

Bovine spermatozoa were incubated in vitro with lysophosphatidylserine (LPS), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), or trypsin. Capacitation of sperm was evaluated by penetration of the zonae pellucidae of dead bovine oocytes. Capacitation times could be shortened to 3 h or less by treatment of spermatozoa with each of these lysophospholipids (LPLs) (P < .05). The maximum oocyte penetration percentages for individual LPLs were 40% for 10 μM LPS, 24% for 160 μM LPC, 31% for 320 μM LPE, and 19% for 320 μM LPI. Capacitation also was facilitated (P < .01) by trypsin treatment of spermatozoa. Spermatozoa treated with 250 or 2,500 units/ml of trypsin penetrated more oocytes (17 and 18%) than spermatozoa treated with 0 or 25 units/ml of trypsin (0 and 3%). Spermatozoa treated with increasing concentrations of LPL showed a decrease in both the percentage of intact acrosomes and of progressively motile spermatozoa. Increasing levels of trypsin in the incubation medium also led to a decrease (P < .05) in the percentages of intact acrosomes and a decrease (P < .01) in the percentages of progressively motile spermatozoa. Percentages of live, ovulated oocytes fertilized by spermatozoa incubated for 1 h in LPS (86%, 6/7) were not different from those incubated for 24 h in control medium (71%, 5/7). Percentages of oocytes fertilized with both of these capacitation treatments were higher (P < .05) than for oocytes exposed or killed or uncapacitated sperm. Rapid induction of capacitation and the acrosome reaction can be accomplished by exogenous treatment of bovine sperm with lysophospholipids or trypsin.     

Uptake and metabolism of androgens by bovine spermatozoa

Spermatozoa from bovine ejaculates and cauda epiditymidis were incubated with either tritiated 17 beta-hydroxy-5 alpha-androstane-3-one (DHT) or 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol). Examination of the medium incubations demonstrated metabolic conversion of both DHT and 3 alpha-diol when these steriods were incubated with ejaculated sperm. In addition to this interconversion, the following metabolities were identified: 5 alpha-androstane-3 beta, 17 beta-diol, (3 beta-diol), androsterone and 5 alpha-androstane-3, 17-dione (5 alpha-A-dione). Incubations with cauda spermatozoa showed similar metabolic patterns. Androgen binding was exhibited by both sperm types. Examination of the washed cauda sperm pellet, following incubations with 3 alpha-diol showed that the incubated steroid was the most abundantly bound. DHT and 5 alpha-androst-16-en-3 alpha-ol (delta 16-3 alpha-ol1 were also detected. The major part of the radioactivity bound in the sperm pellet was identified as DHT when this steroid was used as the substrate; the remaining radioactivity consisted of 3 alpha-diol and delta 16-3 alpha-ol. Investigations of ejaculated sperm pellets gave similar results apart from the additional identification of 5 alpha-androst-16-en-3 one (delta 16-3-one) and 5 alpha-androst-16-en-3 beta-ol (delta 16-3 beta-ol (delta 16-3 beta-ol).     

Double freezing of bovine semen

Fertility of bull spermatozoa cryopreserved in large volume by directional freezing technique, thawed, repackaged in straws and refrozen over liquid nitrogen vapor (double freezing, DF) was compared to conventional single freezing in straws (CF). Semen was collected from 6 bulls, 4 of which were selected for the field trial. Each semen collection was split into two parts, one frozen by CF and the other by DF. In vitro semen evaluations included motility (fresh, upon thawing and after 3 h incubation at 37 °C), viability and acrosome integrity. A total of 3610 cows and heifers were randomly inseminated by either CF or DF at about equal numbers. In vitro sperm analysis indicated no difference between CF and directional freezing in large volume and both were superior to DF (P < 0.001). Between-bull variations in fresh semen and in their reaction to CF or DF were apparent. Logistic regression analysis revealed that freezing method, bull, parity and inseminating technician, all had significant effect on pregnancy outcome (P ≤ 0.001 for all). Conception rate (CR) was 32.98% for CF and 28.05% for DF. Only in one bull conception rate by CF was significantly superior to DF (P < 0.05). When divided into heifers, primi- and pluriparous cows, only the difference in CR between the pluriparous cows was significant (P = 0.005). In conclusion, acceptable CR can be achieved by DF technique. These can be improved by selecting suitable bulls. The DF technique can be utilized in storage, sperm sexing and genome resource banking.     

Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa

Cryopreserved spermatozoa from 8 bulls were used to examine the interrelationships among flow cytometric spermatozoal quality assessments and classical semen quality parameters and nonreturn rate estimates of fertility. The integrity of the sperm cell membrane and the functional capacity of the mitochondria were quantified by flow cytometry after concurrent staining with carboxydimethylfluorescein diacetate (CDMFDA), propidium iodide (PI), and rhodamine 123 (R123). For each sample a total of 10,000 stained spermatozoa were simultaneously quantified for the intensity of their green and red fluorescence. Three straws from each bull were each examined initially and following incubation at 37 degrees C for 3 hours to assess the rate of senescence. The proportion of spermatozoa retaining membrane integrity and having functional mitochondria, as determined by CDMFDA and R123 staining, were compared with classical semen quality assessments (sperm motility, acrosomal status, cellular and head morphology, presence of vacuoles/craters and cytoplasmic droplets) and with fertility (nonreturn to estrus rates). For individual ejaculates nonreturn rates, the range was from 61.8 to 78.8%, whereas the cumulative rates of several ejaculates for each bull ranged from 71.3 to 83.5%. The proportion of spermatozoa with functional membranes and mitochondria were positively correlated with the percentage of spermatozoa with normal morphology (r=0.82; P=0.01) and motility after 4 hours of incubation (r=0.78; P=0.02), but not with the estimates of fertility. The actual number of spermatozoa per straw staining with CDMFDA and R123 after 4 hours of incubation at 37 degrees C was correlated with the percentage of spermatozoa with normal morphology (r=0.73; P=0.04). Multiple regression equations indicated that combinations of semen quality measurements could be useful in estimating fertilizing potential.