Extender components and surfactants affect boar sperm function and membrane behavior during cryopreservation

To determine how the individual components of extenders affected boar sperm function and membrane structure and to test a new surfactant's cryoprotective ability, boar sperm were cryopreserved in straws in BF5 extender plus or minus egg yolk plus or minus glycerol plus or minus a surfactant (Orvus ES Paste [OEP] or various concentrations of Pluronic F-127). After thawing, sperm function and fluidity of the isolated head plasma membrane (HPM) were determined. Total motility and adenosine triphosphate content (a measure of viability) were superior postthaw in sperm extended in egg yolk plus glycerol (P < 0.05); neither surfactant improved function. Egg yolk plus any other ingredients improved normal acrosome morphology, whereas a combined measure of motility and normal acrosome morphology was better in the presence of 0.33% OEP or 0.1% Pluronic F-127 (P < 0.05 vs. controls). Head plasma membrane was isolated from freshly collected spermatozoa and spermatozoa cryopreserved in the various extenders. Membrane fluidity was monitored with the probes cis-parinaric acid (cPNA), transparinaric acid (tPNA), and 1,6-diphenyl-1 ,3,5-hexatriene (DPH). The cPNA and the DPH monitor the fluidity of gel and liquid-crystalline areas of the membrane, whereas the tPNA preferentially monitors the gel-phase domains of the membrane. Additionally, DPH monitors the hydrophobic core of the bilayer. In the HPM from fresh sperm, the fluidity of each domain changed over time in a manner unique to that domain, and the behavior of the DPH domain varied among boars. The fluidity dynamics of each domain responded uniquely to cryopreservation. The cPNA domain was unaffected, the tPNA domain was altered by four of the eight extenders, and all extenders affected the fluidity of the DPH domain. Membrane structure was significantly correlated with cell function for sperm cryopreserved in extenders that preserved viability and motility. Sperm cryopreserved in egg yolk plus glycerol plus either OEP or 0.1% Pluronic F-127 functioned best when the bulk domains were less fluid initially and the gel domain solidified more slowly. Therefore, the behavior of domains in the HPM of boar spermatozoa is affected by cryopreservation and is related to the postthaw function of boar sperm cryopreserved in different extenders.     

Environmental, management, and genetic factors affecting semen production in Holstein bulls

The objective of this study was to evaluate the importance of environment, management, physiological status, and genetics on semen quality (volume of the ejaculate, sperm concentration, sperm motility, number of sperm, and number of motile spermatozoa per ejaculate) of Canadian Holstein bulls. For this purpose, semen production data from 198 bulls were analyzed using mixed linear models. Young bulls (up to 30 mo old) and mature bulls (between 4 and 6 yr old) were analyzed separately. Semen characteristics generally improved significantly with age of young bulls. Season significantly affected all semen traits in young bulls but did not significantly affect volume and sperm motility of mature bulls. Performance was better in winter than in summer. The highest numbers of motile spermatozoa per ejaculate were obtained with intervals of at least 4 to 5 d between collections. Although the bull handler and semen collector caused less than 10% of the variance, the collection team significantly affected semen volume, number of sperm, and number of motile sperm per ejaculate for both growing and mature bulls. Heritabilities for volume, concentration, sperm motility, number of sperm, and number of motile sperm per ejaculate were, respectively, 0.24, 0.52, 0.31, 0.38, and 0.49 for young bulls and 0.44, 0.36, 0.01, 0.54, and 0.64 for mature bulls. Repeatability of semen traits varied from 0.41 to 0.64. Genetics, management, and environmental factors clearly contribute to semen production in Holstein bulls.     

Escherichia coli and Lactobacillus plantarum responses to osmotic stress

Artificial insemination using cryopreserved semen is a common management tool of the contemporary livestock producer. However, cryopreservation is detrimental to sperm function and fertility, killing some 50% of the spermatozoa during the process. Prediction of cryopreservation damage from prefreeze samples remains elusive. Computer-automated sperm head morphometry was used in this study to determine the effects of cryopreservation on bovine sperm head morphometry. Semen was collected from 18 bulls and was divided. One portion was extended to 200 x 10(6) sperm/ml and a microscope slide was prepared, while the remaining portion was cryopreserved in a Triscitrate-yolk extender. After thawing, the cryopreserved samples were prepared on microscope slides. All slides were air dried and were stained with hematoxylin and rose bengal. The morphometric dimensions for length, width, width/length, area, and perimeter for a minimum of 200 sperm heads were analyzed from each slide by computer-aided sperm head morphometry analysis, and the mean measurements were recorded. Bull sperm heads were significantly (P < 0.01) smaller in cryopreserved spermatozoa than in the companion extended samples for length (8.56+/-0.07 vs. 8.63+/-0.08 microm), width (4.39+/-0.05 vs. 4.48+/-0.05 microm), area (28.42+/-0.07 vs. 29.14+/-0.08 microm), and perimeter (23.33+/-0.21 vs. 23.70+/-0.23 microm) for all bulls. Width/length was also different (0.513 vs. 0.519). In addition, differences (P < 0.05) were found within 14 of 18 bulls for at least four of the morphometric parameters. The percent change in measures after cryopreservation were correlated (P < or = 0.05) to the variability of the extended sample. Variations in sperm head measurements were lower (P < or = 0.05) in extended samples of the four bulls in which no changes occurred than in extended samples of the remaining 14 bulls. These data suggest that the variability in sperm head measurements of individual bulls, or ejaculates, may be an indicator of sperm cryosurvivability.     

Reactive oxygen species generation by seminal cells during cryopreservation

OBJECTIVES: To test the hypothesis that conventionally used procedures for semen cryopreservation may cause an increase in the production of reactive oxygen species (ROS) by sperm or by seminal leukocytes, which may contribute to poor sperm function following cryopreservation. METHODS: Eighteen semen specimens with normal parameters from healthy male donors 22 to 40 years of age were each divided into two portions. The first portion was combined 1:1 with Test Yolk Buffer-Glycerol Freezing Medium and was frozen by gradual cooling into liquid nitrogen (-196 degrees C). The second portion was washed and the cells were resuspended in Sperm Washing Medium (SWM) and incubated at room temperature to serve as controls. After a period of treatment, frozen samples were thawed and semen cells were washed and resuspended in SWM. ROS generation by semen cells from each treatment group was measured on a luminometer. Sperm motility, sperm viability, and sperm membrane integrity were also measured in both control and freeze-thaw samples. To further assess ROS generation by semen cells during the cooling process, aliquots of washed semen cells and purified polymorphonuclear leukocytes (PMNs) were incubated separately at different temperature conditions (37 degrees C, 22 degrees C, 4 degrees C, and -20 degrees C). ROS activity in each treatment group was measured and compared with each other. RESULTS: In both semen cells and PMNs, ROS activity increased significantly during the cooling process. The highest ROS levels were recorded in both groups when cooled to 4 degrees C. The ROS levels were extremely low in samples cooled to -20 degrees C and in freeze-thaw samples, probably due to marked loss of cell viability. CONCLUSIONS: Gradual reduction of temperature during the process of semen cryopreservation can cause a significant ROS generation by semen cells. ROS is particularly elevated during cooling if the semen sample is contaminated by more than 0.5 x 10(6) leukocytes. Removal of leukocytes from semen samples or treatment with antioxidants prior to cryopreservation may improve sperm viability and function.     

Outcome of ''whiplash'' neck injury

In this study, the relationships between semen production and semen quality of Normande bulls in performance test station and in two IA centers were evaluated and the genetic characteristics and evolution of semen production of young bulls in station were calculated. When analysing the relationship between semen production of young and adult Normande bulls data from 2,677 bulls between 12 and 15 months of age exhibited large difference between 5 groups corresponding to an overall classification given by a technician. Correlations between corrected data collected in station and bull effect estimates in AZ studs were quite high for volume and concentration, and moderate for motility and number of doses per ejaculate or per month. The correlations calculated are underestimates of the true ones, as bulls with undesirable semen characteristics were culled before entering AI. Nevertheless, among the remaining bulls, important differences (40%) in semen output were observed between extreme groups, as classified by the technician. Genetic parameters evaluated from a subset of 1957 young Normande bulls confirms earlier results reported in the literature. The heritability of the average volume of the ejaculate is very high (h2 = 0.65 +/- 0.09). Sperm quality traits are moderately heritable (h2 = 0.23 +/- 0.08 for motility score to 0.37 +/- 0.09 for concentration) and strongly correlated. Total percentage of abnormal spermatozoa estimated from a selected subset seems less heritable (h2 = 0.19 +/- 0.07). A Best Linear Unbiased Prediction was then carried out on the records from 2,387 bulls using these estimates. No clear genetic trend could be detected in the population, although the phenotypic selection performed should be efficient. This apparent contradiction results from the increasing influence in the breed of one family of heavily used bull sires with a poor genetic merit on sperm production traits.     

Glaxo/MRS Young Investigator Medal. Molecular studies on adenosine deaminase deficiency and hereditary haemorrhagic telangiectasia

Factorially arranged experiments were designed to study prefreeze packed cell volume (PCV) changes and associated percentages of motile and unstained bull sperm in simple macromolecule-free Tyrode's solution and egg yolk-Tris (EYT), varying in osmolarity, and with addition of rapidly permeating cryoprotectants, glycerol and 1,2-propanediol, and nonpermeating substances, sucrose and NaCl. The percentage of motile and unstained sperm was assessed after resuspending sperm in 300 mOsm/L Tyrode's solution. At 25 degreesC PCV increased in Tyrode's solution as osmolarity was decreased from 250 to 150 mOsm/L and decreased as Tyrode's solution was increased to 400 mosmol/L. The relationship of PCV to the reciprocal of the osmolarity was essentially linear over the range of 150 to 400 mOsm/L, but PCV did not decrease further in solutions ranging from 500 to 1000 mOsm/L. The percentage of motile sperm declined to zero in Tyrode's solution at 700 mOsm/L, but 40% of the sperm were still unstained in 1000 mOsm/L solutions. The addition of glycerol or 1,2-propanediol had little effect on PCV. With glycerol or 1,2-propanediol added to 308 mOsm/L Tyrode's solution to give a total of 1267 mOsm/L, there were 49 and 56% motile sperm, respectively, compared to 1% with NaCl added to give 787 mOsm/L. The PCV and percentage of motile sperm suspended in EYT responded to osmotic changes similar to those reported for Tyrode's solution at both 25 and 5 degreesC. Some sperm remained motile after initial exposure to 800 mOsm/L solutions. These findings may have application in improving bull sperm cryopreservation.     

Chromatin structural changes in sperm after scrotal insulation of Holstein bulls

The reported effects on semen quality ascribed to testicular heat stress generally relate to traits impacting sperm transport and fertilizing ability but not to the genetic material contained by the sperm. To characterize the effects of testicular heat stress on sperm chromatin, susceptibility of DNA in sperm nuclear chromatin to in situ acid denaturation was measured by flow cytometry after staining with acridine orange using the sperm chromatin structure assay (SCSA). Semen was collected from Holstein bulls at 3-day intervals, before and after 48-hour scrotal insulation, until the morphologically abnormal sperm content in raw semen exceeded 50%. After cryopreservation in egg yolk-citrate extender, semen was thawed and sampled during incubation in vitro at 38.5 degrees C. Overall, SCSA results showed that chromatin susceptibility to denaturation was increased for sperm collected post- vs. preinsulation and was more pronounced for sperm presumably in the testes during insulation than for those sperm presumably in the epididymides. Increased susceptibility was detected as early as the first collection postinsulation; however, chromatin of sperm presumably in the proximal epididymis during insulation did not appear to have been detrimentally affected. Chromatin susceptibility to denaturation increased with increased incubation time in vitro, but the rate of change in susceptibility during incubation did not differ among pre- vs. postinsulation specimens. We conclude that elevated scrotal temperatures adversely affect both epididymal and testicular sperm by reducing sperm chromatin stability. The effects of heat stress on the chromatin of epididymal sperm were more subtle than those exhibited by testicular sperm but detectable within close proximity to the heat stress event.     

Flow cytometric and microscopic evaluation and effect on fertility of abnormal chromatin condensation in bovine sperm nuclei

The techniques of Feulgen staining, acridine orange staining, and a sperm chromatin structure assay using acridine orange and flow cytometry were compared for selective examination of bovine sperm nuclei. Twenty frozen semen samples were simultaneously analysed by all three methods. The prevalence of abnormally condensed DNA and its relationship to other semen traits were determined in ejaculates from 70 bulbs presented for routine examination for breeding soundness and in frozen semen from 348 bulls evaluated over five years. A breeding trial with 118 beef heifers using semen from six bulls with different degrees of nuclear abnormalities was performed to assess the importance of the defects with respect to fertility. The results indicate that few spermatozoa with abnormal DNA condensation are found in normal semen, but the incidence increases with disturbance of spermatogenesis. However, high numbers of abnormally condensed nuclei were found in the absence of an increase in other defects. This nuclear defect might be at least partially of epididymal origin; it can lower fertility and can be compensated for by increasing the numbers of normal spermatozoa in the insemination dose. The percentage of abnormally condensed sperm nuclei as detected by Feulgen staining was significantly correlated with that detected by microscopy after acridine orange staining and by the sperm chromatin structure assay. We therefore consider the Feulgen technique to be a valuable tool for assessing the nuclear integrity of bovine spermatozoa.     

Enhancement of motility by treating spermatozoa with an antioxidant solution (Sperm-Fit) following ejaculation

Oxygen radical generation is known to be detrimental to sperm function, especially motility, through the lipid peroxidation of the membranes. Generation of reactive oxygen species can be induced by leukocyte contamination, sperm centrifugation and the presence of abnormal spermatozoa with excess residual cytoplasm. This study aims to evaluate the effect on sperm motility of incubation in an antioxidant-containing solution, during liquefaction and centrifugation. Thirty semen samples were each divided into two equal parts: one mixed with Tyrode's solution, the other with a salt solution containing antioxidants (Sperm-Fit; Ellios Bio-Media, Paris, France). All the procedures were identical in the two groups. The ratio of leukocytes to spermatozoa was significantly correlated with the motility after liquefaction and after a 24 h incubation in routine in-vitro fertilization (IVF) medium and with the number of motile spermatozoa recovered after Percoll preparation. Moreover, when this ratio was > or = 0.2, all motility parameters were lowered. Incubation with Sperm-Fit allowed a higher percentage of motility after Percoll preparation when the ratio was > or = 0.2 (48 +/- 5% versus 41 +/- 6% for Sperm-Fit and Tyrode's solution respectively; P < 0.05) and a greater number of motile spermatozoa recovered after Percoll preparation, whatever the ratio (3.2 +/- 1.0 x 10(6) versus 2.4 +/- 0.7 x 10(6) for Sperm-Fit and Tyrode's solution respectively when ratio > or = 0.2; 18.1 +/- 3.4 x 10(6) versus 14.4 +/- 2.9 x 10(6) for Sperm-Fit and Tyrode's solution respectively when ratio < 0.2; P < 0.05). These results show that incubation with antioxidants during liquefaction and centrifugation increases recovery of motile spermatozoa.     

Studies on the development of diluents for the transportation and storage of human semen at ambient temperature

Storage of human semen samples at ambient temperature for 24 h resulted in a significant loss of sperm motility from a mean 45.1 +/- 1.8% to 13.8 +/- 1.1% (n = 148). This motility loss was associated with a significant increase in the osmolality of the seminal plasma and the induction of peroxidative damage to the spermatozoa. Both of these detrimental changes could be prevented by diluting the original semen sample 1:1 with a citrate-egg yolk, buffer (CYB). In the presence of this extender all aspects of semen quality were efficiently preserved for 24 h, including sperm movement, penetration of a cervical mucus substitute, the acrosome reaction and sperm-oocyte fusion. CYB extension also permitted the use of chemiluminescent tests of leukocyte contamination to be performed on semen samples stored for 24 h at ambient temperatures. As a preservation medium, CYB was found to be superior to alternative formulations lacking citrate and storage at ambient temperatures was preferable to 4 degrees C. Significant improvements in motility retention were also observed when CYB was supplemented with pentoxifylline, although this treatment significantly stimulated peroxidative damage in the spermatozoa. However, if the pentoxifylline was combined with antioxidants then this collateral peroxidative damage could be reduced and the performance of CYB significantly enhanced. These results have implications for the design of diluents permitting the long-term storage and transportation of human semen samples at ambient temperatures.     

Modulation of postthaw motility, survival, calcium uptake, and fertility of bovine sperm by magnesium and manganese

Because Mg2+ and Mn2+ are potent stimulators of motility through the stimulation of adenylate cyclase activity, the current study was undertaken to modulate the fertilizing ability of bovine semen by incorporation of various concentrations of those two salts in extenders before freezing. Motility analysis at 6 h in vitro showed a positive effect of MgCl2 in a dose-dependent manner from 0.5 to 5 mM (31 to 50%). Manganese at the concentration of 0.1 mM also supported good sperm motility (53%) compared with that of the control (28%). Although survival was increased, no detrimental effects were seen on the number of sperm that penetrated mucus of cows in estrus. The intracellular Ca2+ concentration of sperm was very different across treatments after thawing; spermatozoa that were extended with 2 mM MgCl2 and 0.5 mM MnCl2 possessed the highest concentrations at thawing. Four hours later, in the presence of Ca, spermatozoa that were extended in 0.1 mM MnCl2 showed the highest uptake. In the presence of Ca and heparin, spermatozoa that were extended in different amounts of Mg showed Ca2+ concentrations that increased in a dose-dependent manner. This effect was negated by glucose. Functional fertilizing capacity was also evaluated by in vitro fertilization, and the different treatments did not show any detrimental effects. In summary, 5 mM MgCl2 and 0.1 mM MnCl2 both have beneficial effects for the maintenance of sperm motility without detrimental effects on mucus penetration and fertilizing ability. Furthermore, these treatments do not prevent subsequent Ca2+ uptake in response to heparin. These in vitro studies are potentially a good sorting system to predict the benefits of extender modifications.     

Effect of follicular or oviductal fluids on movement characteristics of bovine sperm during capacitation in vitro

Mammalian sperm exhibit characteristic motility changes associated with capacitation. Movement characteristics of bovine sperm incubated in noncapacitating (control, medium alone), capacitating (oviduct fluid, nonluteal, and luteal), or capacitating, acrosome reaction inducing (follicular fluid) conditions were investigated using a computer-assisted automated semen analysis system. Sperm were incubated up to 4 hours in a modified Tyrode's medium (control), 20 and 60% nonluteal (NL) or luteal (L) oviduct fluid (ODF), or 20 and 60% follicular fluid (FF). Relative to sperm incubated in control medium, motility of sperm treated with ODF or FF had increased linearity and vigorous motility. Sperm incubated in 60% ODF or FF showed a small decrease in mean trajectory/path straightness and velocity over time compared to 20% fluid treatments and control. Frequency distribution graphs were symmetric for 20% NL- and L-ODF treated sperm. However, 20% FF and 60% ODF and FF treatments had distributions skewed to the left, indicating smaller values for lateral head displacement (ALH) and curvilinear velocity (VCL). Median values for ALH and VCL were determined for control-treated sperm, and subtracted from individual sperm values for all treatments to estimate deviation from control, designated ALHc and VCLc. Three-dimensional plots of ALHc, VCLc and corresponding frequency indicated shifts in peak patterns for fluid-treated sperm compared to control sperm. Incubation in 20% ODF and FF resulted in peak shift for ALH and VCL values; yet, little change in peak position was observed in sperm incubated in 60% ODF and FF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depot medroxyprogesterone acetate. Patterns of use and reasons for discontinuation

In some countries, sperm counts in normal human semen seem to have declined over the last 50 years. If this decline is real and due to environmental factors, falls might also be seen in sperm numbers in the semen of farm animals. Sperm counts are available for bull, boar and ram from the early 1930s, obtained using techniques similar to those used for human semen. Data have been obtained from the literature between 1932 and 1995 from 137 studies involving bulls, 76 involving boars and 130 involving rams. All were normal adult animals, from which semen was collected regularly but at a frequency which would not be likely to cause a fall in sperm counts. The references were obtained systematically from Animal Breeding Abstracts, and where possible the original articles were consulted to obtain mean values for each study; where the original reference was not easily obtainable, values were taken from the abstract. The bull data showed no correlation of sperm count with year of publication (r2 = 0.000), for the boars there was a slight but non-significant positive correlation (r2 = 0.041), and for the sheep there was a slight, but significant, rise in sperm counts with time (r2 = 0.124 for sperm counts and 0.126 for total sperm per ejaculate; not all authors gave both values). It would appear that, if the fall in human sperm counts is real, then it must be due to something which is not affecting farm animals.     

In vitro maturation and fertilization techniques for assessment of semen quality and boar fertility

The reliability of using different in vitro-derived measures of sperm quality to predict boar fertility was examined. On three occasions during a 20-wk period of breeding, special collections of the first sperm-rich fraction of the ejaculate from six boars were carried out. After in vitro capacitation procedures, three dilutions (5 x 10(5), 1.25 x 10(5), and 3.125 x 10(4) sperm/mL) of these semen samples were used in a standardized in vitro fertilization (IVF) test with oocytes recovered from prepubertal slaughterhouse ovaries and matured in vitro. Routine assessments of sperm motility, concentration, and morphology were also carried out for all collections used for AI during the 20-wk period. Semen from the same ejaculate, processed according to normal commercial practice using the AndroHEP extender, was used to inseminate equal numbers of recently weaned sows with either 3 x 10(9) or 2 x 10(9) total sperm, three times during the estrous period. Data from a total of 444 sows were used to determine boar fertility; between 12 and 54 sows were bred with each semen dose across the six boars. All measures of sperm fertilizing ability in vitro were different among boars (all P < .05) and use of different semen dilutions for IVF allowed further discrimination of apparent sperm quality among boars. The laboratory evaluation of semen collected during the period of breeding indicated effects of boar on ejaculate volume, total number of sperm per ejaculate, motility, and the percentage of sperm with normal morphology (all P < .01). Sperm dose used in AI had no effect on farrowing rate (80.7 vs 81.5%), but the lower AI dose resulted in a reduction (P < .05) in total numbers born (10.8 vs 10.0). For all three semen dilutions, estimated potential embryo production rate accounted for up to 70% of the variation in litter size obtained with 3 x 10(9) sperm per AI dose, and the number of sperm attached per oocyte was a major factor accounting for variation in litter size obtained with 2 x 10(9) sperm per AI dose. These IVF variables may, therefore, be effective indicators of boar sperm quality for use in AI. With 2 x 10(9) sperm per AI dose, the percentage of sperm with normal morphology also explained a large part of the variance in litter size born (R2 = .59), indicating that morphological characteristics are a useful measure of semen quality.     

Biochemical properties and usefulness of boar semen for liquid preservation following atropine administration

Four sexually mature boars were used in two different experiments. In Experiment I, two boars were injected once subcutaneously with atropine and, ejaculates were collected after 30, 60 and 90 min. In Experiment II, each boar was injected weekly with 25, 37.5 and 50 mg of atropine, and ejaculates were collected after 30 min. Ejaculates obtained in Exp. II were diluted with Kortowo-3 extender (Olsztyn, Poland), with and without the addition of low density lipoprotein fraction (LDF) isolated from hen egg yolk,and stored at 5 degrees C and 16 degrees C for 5-6 days. Atropine caused a decrease in semen volume and an increase in sperm concentration with fewer agglutinated spermatozoa. Changes in sperm motility were not significant. There was a significant increase in the content of fructose, zinc, citric acid and protein in the seminal plasma of atropine-injected boars. Furthermore, increased antiproteolytic and antiperoxidant activity as well as seminal phosphatases were also observed. No significant changes were observed in the content of free sialic acid, whereas bound sialic acid was significantly increased in Exp. II. A decrease in osmolality and pH of seminal plasma was observed. Electrophoretic studies revealed that there were alterations in the molecular forms of seminal phosphatases and proteinase inhibitors. There were no significant changes in the percentage of morphologically abnormal spermatozoa, osmotic plasmalemma resistance at the acrosomal region (ORT) and malondialdehyde production in the spermatozoa. AspAT activity recovered from cold shocked spermatozoa was significantly reduced, whereas disturbances in plasma membrane permeability to fluorochrome HO 258 were observed in Exp. I. Semen of atropine-injected boars had increased sperm viability during liquid preservation at 5 and 16 degrees C.     

Fertility of bull semen frozen with beta-amylase, beta-glucuronidase, and catalase

"Capacitase," a product combining beta-amylase and beta-glucuronidase, was compatible with survival of bull spermatozoa frozen in whole milk-glycerol extender at final concentrations per ml of 0, 5, 10, and 20 mug of beta-amylase combined with 0, 75, 150, and 300 units of beta-glucuronidase, respectively. Bull semen was frozen in whole milk-glycerol extender containing the three lower concentrations of enzymes tested in the previous trial and used to inseminate 9057 first-service cows within 4 mo of freezing. The 60- to 90-day percent nonreturns were 74.6, 75.6, and 75.0. The same treatments plus a fourth one containing 10 mug of catalase per ml were fertility tested in another trial. Insemination of 16,842 cows resulted in 75.6, 74.1, 74.6, and 74.2% nonreturns. In this trial semen was held immersed in liquid nitrogen and distributed for immediate use each mo for 6 mo. There was no change in fertility during 6 mo of continuous storage at --196 C. Under the conditions tested neither catalase nor beta-amylase with beta-glucuronidase enhanced fertility of frozen bull semen.     

Comparison of sperm separation methods: effect on recovery, motility, motion parameters, and hyperactivation

OBJECTIVE: To compare the Enhance (Percoll; Conception Technologies, San Diego, CA) and PureSperm (Gen X International, Madison, CT) sperm preparation methods with respect to recovery (percentage of motile sperm), motility (%), path and progressive velocities (microm/s), and hyperactivation (%). DESIGN: Comparison of sperm processing methods. SETTING: University medical center-based clinical andrology laboratory and infertility program. PATIENT(S): Twenty-five men who presented for semen analysis. INTERVENTION(S): Each of 25 semen specimens were divided and each aliquot was prepared using two different density gradient centrifugation methods. MAIN OUTCOME MEASURE(S): The motile sperm recovery, percent motility, motion parameters, and percent hyperactivation were measured for each semen specimen (n=25) before and after separation with the use of the two methods. RESULT(S): There was no difference in the percent motility and motile count between specimens prepared with Enhance (Percoll) and PureSperm and fresh specimens. Statistically significant differences were found (fresh versus test) in the velocities and in hyperactivation (PureSperm only), and no differences were found between the processing methods. CONCLUSION(S): PureSperm appears to be as effective as Percoll (Enhance) for the recovery of good, progressively motile sperm for use in IUI or other assisted reproductive techniques.     

Sperm numbers inseminated in dairy cattle and nonreturn rates revisited

Three experiments were conducted to test fertility when sperm numbers per insemination ranged from 10 x 10(6) to 40 x 10(6) total sperm. All semen was from Holstein bulls that were on a regular schedule of semen collection. The semen was extended with heated homogenized whole milk, cooled, glycerolated, and frozen according to standard procedures. Semen was distributed to a large group of inseminators to minimize differential field effects on treatment. All experiments were a randomized block design, including a split plot in Experiment 2. In Experiment 1, data for 31,399 first inseminations distributed among treatments of 20 x 10(6), 25 x 10(6), 30 x 10(6), and 40 x 10(6) total sperm resulted in 69.8, 70.0, 70.1, and 70.1% nonreturns at 59 d, respectively. In Experiment 2, data for 18,197 first inseminations divided over treatments of 12 x 10(6), 16 x 10(6), and 20 x 10(6) total sperm resulted in 70.2, 72.4, and 70.8% nonreturns at 59 d, respectively. In Experiment 3, 38,890 first inseminations distributed over treatments of 10 x 10(6), 13 x 10(6), 16 x 10(6), and 20 x 10(6) total sperm resulted in 70.5, 72.2, 73.1, and 71.5% nonreturns at 59 d, respectively. Bull nonreturns ranged from 64 to 76% in the three trials. These results indicate that, under good conditions, total sperm numbers per straw can be reduced to 10 x 10(6) total sperm with a reduction of nonreturn rates at 59 d, for most bulls, of about 1 percentage unit from the maximum when professional inseminators are use.     

Manual collection and characterization of semen from Asian elephants (Elephas maximus)

The implications of collecting semen from elephants for use in artificial insemination programs are profound in the context of propagating captive elephants. Using a manual manipulation technique, semen was collected and characterized from five adult Asian elephants (Elephas maximus) and ejaculate fluid was obtained from one castrated elephant bull. The penis was stimulated to protrusion and erection by rectal massage of the pelvic portion of the urethra. During an ejaculatory response, massage was also directed onto the area of the ampulla of the ductus deferens. Sperm rich ejaculates were usually collected as a result of each ejaculatory contraction. Ejaculates were evaluated for spermatozoal concentration and pH (when possible) and sperm rich fractions combined for determination of total volume. Mean total volume of each collection was 27.5+/-4.4 ml. Mean concentration of the first and second ejaculatory responses from a collection was 2.05+/-0.17 x 10(9) and 1.34+/-0.19 x 10(9) sperm/ml, respectively. Measurement of seminal pH revealed no significant differences between the fractions. Mean pH of the first and second ejaculatory responses were 7.05+/-0.07 and 7.04+/-0.13. This method of collecting elephant sperm can be utilized for semen evaluation of bulls of unknown reproductive status in conjunction with other evaluation techniques (i.e. ultrasonographic, endocrinologic). It also has the potential for providing valuable genetic material for genome resource banks and for use with assisted reproductive techniques like artificial insemination.     

Rotational digital subtraction carotid angiography: technique and comparison with static digital subtraction angiography

To compare the efficiency of sperm preparation between the two-layer Percoll gradient and mini-Percoll methods, 50 normal and 33 abnormal semen samples from male partners of infertile couples were studied. The number of recovered spermatozoa, percentage of motility, percentage of normal morphology, and their survival at 24 and 48 hours were assessed. Both Percoll gradient techniques resulted in a significantly higher percentage of motility and percentage of normal morphology compared with the original semen samples (p < 0.0001). The two-layer Percoll gradient showed a higher sperm recovery than the mini-Percoll method (p < 0.001), but the latter resulted in a higher percentage of motility (p > 0.001) and a higher sperm survival rate at 24 hours (p < 0.05) than the former, regarding normal semen samples. These differences did not appear with abnormal semen samples when analyzed as a group. Considering each of the abnormal parameters separately, sperm recovery was significantly higher after the two-layer Percoll gradient in the case of astheno- and teratozoospermia (p < 0.05), but sperm survival at 48 hours was higher after the mini-Percoll gradient in the case of teratozoospermia (p < 0.05). It is concluded that both the two-layer Percoll gradient and mini-Percoll method can be used effectively for sperm preparation. The former yields a higher sperm recovery, but the latter should be considered regarding teratozoospermic samples and semen samples of very low volume.