Assessment of goat semen freezability according to the spermatozoa characteristics from fresh and frozen samples

A total of 110 ejaculates were assessed in order to determine the influence of the physical parameters of goat semen on post-thaw motility and acrosome integrity. Sperm ejaculate characteristics, sperm motility, morphology and acrosome integrity were assessed in fresh and frozen samples by the sperm class analyzer (SCA) and Spermac staining technique. A decrease in acrosome integrity and sperm motility was found after thawing (P<0.01). Six semen parameters assessed before freezing were identified as predictors of sperm freezability (P<0.01). The percentage of morphological abnormalities (R=0.856) and motile sperm cells (R=0.655) in fresh semen are the best predictors to know the total post-thaw variability parameters.     

Sperm morphology is better in the second ejaculate than in the first in domestic cats electroejaculated twice during the same period of anesthesia

Several species produce ejaculates of inferior quality after a period of sexual abstinence, but the frequency of semen collection has thus far not been shown to affect sperm morphology in felids. The aim of this study was to determine whether sperm morphology and motility would differ between 2 ejaculates collected from the same cat within a short interval. Fifteen male domestic cats were anesthetized and then electroejaculated twice, with a 5- to 10-min interval between treatments. A standardized electroejaculation regimen was used with 80 stimuli, from 2 to 5 V, for each ejaculate. The first ejaculates contained significantly higher (P < 0.05) proportions of distal droplets, coiled tails and immotile spermatozoa than the second ejaculates, which contained significantly higher proportions of morphologically normal spermatozoa (40.9 vs 54.6%) but a lower sperm count (39.0 x 10(6) vs 5.2 x 10(6)). The higher proportions of defective spermatozoa and the lower motility in the first ejaculate than in the second were probably due to the aging of spermatozoa in the epididymis. These results show that the second ejaculate collected within a short interval has better sperm morphology and motility than the first and that this should be considered when evaluating semen quality in the domestic cat and when collecting cat semen to be used for artificial insemination or to be frozen for storage.     

Semen characteristics of the swamp buffalo (Bubalus bubalis)

The semen characteristics were studied in 182 ejaculates collected with a bovine artificial vagina from five swamp buffalo (Bubalus bubalis) bulls. The mean values were: volume, 2.9 ml; general motility, 70.7%; live (unstained) sperm, 86.5%; abnormal sperm, 10.3%; intact acrosomes, 82.4%; sperm concentration, 1.06 × 10⁹cells/ml and total sperm/ejaculate, 3.18 × 10⁹cells/ml. Among the sperm abnormalities noted were “knobbed” acrosome, abaxial implantation, the “Dag” defect and the corkscrew midpiece. There were no significant (P > 0.05) monthly variations for any of the semen characteristics studied.     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.     

Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions

This study analyzed effects of different methods and intervals of semen collection on the quantity and quality of fresh, cool-stored, and frozen-thawed sperm and fertility of AI stallions. In Experiment 1, ejaculates were obtained from six stallions (72 ejaculates per stallion) using fractionated versus non-fractionated semen collection techniques. Initial sperm quality of the first three jets of the ejaculate was not different from that of total ejaculates. Centrifugation of sperm-rich fractions before freezing improved post-thaw motility and sperm membrane integrity when compared to non-centrifuged sperm-rich fractions or non-fractionated centrifuged ejaculates (P<0.05). In Experiment 2, semen from four stallions (60-70 ejaculates per stallion) was collected either once daily or two times 1h apart every 48 h. The first ejaculates of double collections had significantly higher sperm concentrations, percentages of progressively motile sperm (PMS) after storage for 24h at 5 degrees C and lower percentages of midpiece alterations than single daily ejaculates. Semen collected once daily showed significantly lower values of live sperm after freezing and thawing than the first ejaculate of two ejaculates collected 1h apart every 48 h. In Experiment 3, semen was collected from 36 stallions (> or =12 ejaculates per stallion) during the non-breeding season and the time to ejaculation and the number of mounts was recorded. When time to ejaculation and the number of mounts increased, volume and total sperm count (TSC) also increased (P<0.05), whereas a decrease was observed in sperm concentration, percentage of PMS after storage for 24 h at 5 degrees C, percentage of membrane-intact sperm in fresh semen (P<0.05) as well as motility and percentage of membrane-intact sperm of frozen-thawed sperm (P<0.05). In Experiment 4, AI data of 71 stallions were retrospectively analyzed for the effect of number of mounts per ejaculation and frequency, time interval of semen collections on pregnancy, and foaling rates (FRs) of mares. Semen volume increased, but sperm concentration and percentage of PMS after 24-h cool-storage decreased with increasing number of mounts on the phantom (P<0.05). A statistically significant inter-relationship was demonstrated between frequency and interval of semen collection and FR. Mares inseminated with stallions from which semen was collected frequently (> or =1 on an average per day) showed significantly higher FRs than mares inseminated with semen from stallions with a daily collection frequency of 0.5-1 or <0.5. FR of mares inseminated with stallions having 0.5-1 days between semen collections was significantly better than FR of mares that were inseminated with stallions having semen collection intervals of 1-1.5 days or >2.5 days.     

Amino acid changes within conserved region III of the herpes simplex virus and human cytomegalovirus DNA polymerases confer resistance to 4-oxo-dihydroquinolines,a novel class of herpesvirus antiviral agents

The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.     

Influence of ejaculation frequency of stallions on characteristics of semen and output of spermatozoa.

Approximately 1 week was required to stabilize the extragonadal sperm reserves in stallions ejaculated daily for 10 weeks. The true daily sperm output of a stallion was equal to the mean daily sperm output of seven ejaculates +/- 1-35 X 10(9) spermatozoa. Mean concentrations of spermatozoa/ml and number of spermatozoa/ejaculate were higher (P less than 0-01) for X1 and X3/week ejaculation frequencies than for a X6/week frequency. Sperm output/week was nearly identical for a X6/week frequency. Sperm output/week was nearly identical for the X3 and X6 frequencies and higher (P less than 0-01) than the X1 frequency. Increase of ejaculation frequency from one to two ejaculates/day twice weekly significantly (P less than 0-01) raised the output of spermatozoa/week. Gel-free semen volume, spermatozoa/ml, and number of spermatozoa/ejaculate were higher (P less than 0-01) in the first, than in the second, ejaculate. Collection of semen on alternate days would be a practical ejaculation frequency for inseminating mares. Two ejaculates collected twice a week would be a practical ejaculation frequency for long-term storage of stallion semen.     

Electroejaculation, semen characteristics and serum testosterone concentrations of free-ranging african elephants (Loxodonta africana)

A regimented electroejaculation protocol (120 electrical stimulations; 10-30 V) was used to collect semen and characterize ejaculate quality from 9 adult, free-ranging African elephants under anaesthesia. Eight of the 9 ejaculates contained high concentrations of progressively motile spermatozoa. The overall mean ejaculate volume, sperm concentration/ml ejaculate, sperm motility, sperm status and ejaculate pH were 93.3 ml, 2408.6 X 10(6) spermatozoa/ml, 70%, 3.9 and 7.4, respectively. A high percentage (mean 77.5%) of spermatozoa within each ejaculate was morphologically normal. Of the aberrant spermatozoa, 72% had a cytoplasmic droplet defect. When sperm viability was tested in vitro at 37 degrees C, sperm motility rating declined by at least half of the initial assessment within 3.5 h of semen collection. Generally, spermatozoa maintained motility in vitro for less than 6 h. Serum testosterone ranged from 1.4 to 8.2 ng/ml in 4 males evaluated in the morning (07:30-08:00 h). In 4 of the 5 bulls assessed in the afternoon (15:00-18:00 h), testosterone levels were less than 0.9 ng/ml. The remaining bull, evaluated at 16:00 h, had exceptionally high testosterone concentrations (peak 25.6 ng/ml) and a preputial discharge potentially indicative of 'musth'. The present study demonstrates that high quality semen can be collected consistently from the African elephant and that striking differences exist in serum testosterone amongst free-ranging males which may be due, in part, to a diurnal rhythm.     

Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry

Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ejaculate per stallion were thawed at 37 degrees C for 30s. Motility was analysed with CASA immediately after thawing, while for flow cytometry spermatozoa were cleansed by 70:40% Percoll discontinuous density gradient separation before analysed for sperm viability, acrosome integrity (stained with SNARF, PI and FITC-PSA) and capacitation status (stained with Merocyanine 540/Yo-Pro-1). Results (as least square means) were as follows: the motility of frozen-thawed semen was 43.4% for Tori stallions and 42.3% for Estonian stallions (P>0.05). After Percoll separation 79.3% of the spermatozoa from Tori stallions had intact acrosomes and 1.7% of them showed early signs of capacitation. The same parameters for Estonian stallions were 84.5 and 2.3%, respectively. There were no statistically significant differences between breeds or ejaculates within breed for any evaluated parameter. We conclude that triple staining and flow cytometry are valuable techniques to evaluate frozen-thawed stallion spermatozoa, and that no differences in quality of frozen semen were registered between Tori and Estonian breed stallions, allowing implementation of this technology in the Estonian horse population.     

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.     

Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen

The sensitivity of dog sperm cells for extracellular Ca(2+)/Ca(2+)-ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm-rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrode's medium (HBT containing 0.1 mM Ca(2+)), without (control sample) and with 2.5 microM Ca(2+)-ionophore (induced sample) and incubated for 60 min at 38 degrees C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5-ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer-driven three-step freeze protocol and subsequently thawed for 50 sec in a 37 degrees C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein-conjugated peanut agglutinin (PNA-FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD-1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca(2+)-ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca(2+)-ionophore treatment correlated very negatively (r = -0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca(2+)-ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca(2+)-ionophore treatment followed by simultaneous determination PNA-FITC and EthD-1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors.     

Evidence of deteriorating semen quality in the United Kingdom: birth cohort study in 577 men in Scotland over 11 years.

OBJECTIVE: To determine whether the quality of semen has changed in a group of over 500 Scottish men born between 1951 and 1973. DESIGN: Retrospective review of data on semen quality collected in a single laboratory over 11 years and according to World Health Organisation guidelines. SETTING: Programme of gamete biology research funded by Medical Research Council. SUBJECTS: 577 volunteer semen donors. Of these, 171 were born before 1959, 120 were born in 1960-4, 171 in 1965-9, and 115 in 1970-4. MAIN OUTCOME MEASURES: Conventional criteria of semen quality including semen volume (ml), sperm concentration (10(6)/ml), overall motility (% motile), total number of sperm in the ejaculate (10(6)), and total number of motile sperm in the ejaculate (10(6)). RESULTS: When the four birth cohort groups were compared a later year of birth was associated with a lower sperm concentration, a lower total number of sperm in the ejaculate, and a lower number of motile sperm in the ejaculate. The median sperm concentration fell from 98x10(6)/ml among donors born before 1959 to 78x10(6)/ml among donors born after 1970 (P=0.002). The total number of sperm in the ejaculate fell from 301x10(6) to 214x10(6) (P=0.0005), and the total number of motile sperm in the ejaculate fell from 169.7x10(6) to 129.0x10(6) (P=0.0065). CONCLUSION: This study provides direct evidence that semen quality is deteriorating, with a later year of birth being significantly associated with a reduced number of sperm in adult life.     

Effects of age and environmental factors on semen production and semen quality of Austrian Simmental bulls

More than 90% of the breeding stock of Austrian dual purpose Simmental cows is artificially inseminated. Knowledge of factors affecting sperm production and semen quality is of importance with regard to reproductive efficiency and thus genetic improvement as well as for the productivity and profitability of AI centers. Hence, semen data from two Austrian AI centres collected in the years 2000 and 2001 were evaluated. In total, 3625 and 3654 ejaculates from 147 and 127 AI bulls, respectively, were analysed regarding ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. Effects accounted for were the bull (random), age of bull, collection interval, number of collection on collection day, bull handler, semen collector, temperature on day of semen collection, in the course of epididymal maturation (average temperature of days 1-11 before collection) and during spermatogenesis (average temperature of days 12-65 before collection). Age of bull significantly affected all traits (P<0.01 to P<0.001) except motility score in center 2. Ejaculate volume and total number of spermatozoa increased with age of bull while sperm concentration was lower in higher age classes (center 1). The collection team was also found to significantly influence semen quality traits. With increasing collection interval ejaculate volume and total number of spermatozoa increased significantly (P<0.05 to P<0.001) while collection intervals between 4-9 days and 1-6 days were superior with regard to sperm concentration and percentage of viable spermatozoa, respectively (P<0.10 to P<0.001). First ejaculates were superior with respect to ejaculate volumes, sperm concentrations and total number of spermatozoa per ejaculate (P<0.001). Temperature, either on day of semen collection or during epididymal maturation or spermatogenesis, had important but inconsistent effects on semen production and sperm quality. Overall, however, ambient temperatures in the range of 5-15 degrees C were found to be optimal for semen production.     

Sperm factors related to in vitro penetration of porcine oocytes

This study was designed to evaluate the relationship between sperm factors and penetration capacity in an in vitro system with immature porcine oocytes. The sperm parameters evaluated in 145 ejaculates were volume, sperm concentration, total cells in the ejaculate, ATP content, morpho-anomalies, percentage of motile sperm cells, forward progressive motility (FPM), acrosome status (NAR), hypo-osmotic swelling test (HOS), osmotic resistance test (ORT), eosin-nigrosin viability stain and sperm membrane integrity (DCF). Porcine oocytes (a total of 8,736) were used to evaluate the capacity of the different sperm assays to predict penetration. Many parameters were found to be related to in vitro penetration ability; all conventional semen parameters, except sperm concentration and eosinnigrosin staining, were significantly better in high (>75%) than in low penetration rates (<75%). When the ejaculates were preselected the number of significantly related parameters was lower. When studying all conventional semen parameters through a stepwise multiple linear regression analysis of seminal measurements, up to 72.3% of total variance of the penetration rate could be predicted. However, as many as 4 parameters were needed (FPM in fresh semen, folded tail, NAR in post-treatment semen and DCF) for accurate prediction. On the other hand, the multiple logistic regression needed 7 parameters to discriminate 83.96% of the cases correctly. In summary, the results from the present study showed that almost all studied parameters were significantly different for predicting penetration process attained or failed, but most of them were correlated together. These findings emphasize the complexity of sperm functions and the difficulty of assessing the fertilizing ability.     

Objective analysis of stallion sperm motility

An image-analysis system utilizing a microcomputer and CellSoft computer-assisted semen analysis software package was evaluated to assess stallion sperm motility characteristics. Analyses were performed at 37°C on a 6 μl drop of diluted semen placed on a glass slide and covered with an 18 mm² coverslip. Four groups of 25 cells each per slide, four slides per ejaculate and four ejaculates from each of three stallions were analyzed in a nested model. The percentage of motile sperm cells, mean velocity (μm/sec), mean linearity, and mean angular head displacement (μm) were measured. Statistical analysis of variance components showed that within ejaculates, more variation was accounted for in the differences among groups of 25 cells than among slides. Predicted standard deviations calculated for combinations of slides and groups of cells showed that a combination of two slides from which a total of 400 cells were analyzed resulted in a mean intra-assay coefficient of variation (CV) of 5.7% for the four measured variables. The following are individual coefficients of variation: percentage of motile cells (7.8%), mean velocity (6.4%), mean linearity (1.9%) and mean angular head displacement (6.6%). When ejaculate differences were included in the model and predicted standard deviations were calculated for a single ejaculate, the mean inter-assay CV was 9.2%. Mean velocity (6.4%) and mean linearity (4.7%) were more repeatable among ejaculates than either the percentage of motile sperm (14.4%) or angular head displacement (11.2%). It was concluded that this system is precise enough to determine differences in motility characteristics of stallion semen samples.     

Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of semen fractionation and dilution ratio on motility parameters of stallion spermatozoa. In Experiment 1, three ejaculates from each of three stallions were divided into sperm-rich (SR) and sperm-poor (SP) fractions to determine the difference in sperm concentration. Mean sperm concentration in SR fractions (349.5 x 10(6)/ml) was greater (P < 0.001) than that of SP fractions (96.9 x 10(6)/ml). In Experiment 2, three ejaculates from each of two stallions were divided into SR and SP fractions. Fifty percent of the original volume of SR fractions was combined with 50% of the original volume of SP fractions for each ejaculate to represent total ejaculates. SR and total ejaculates were diluted with skim milk-glucose semen extender as follows: 1) no dilution, or dilution to 2) 100 x 10(6)sperm/ml, 3) 50 x 10(6)sperm/ml, or 4) 25 x 10(6)sperm/ml. Semen samples were evaluated at 0.5, 3, 6, 12, and 24 h postejaculation (25 degrees C storage temperature) for percentages of total spermatozoal motility (TSM) and progressive spermatozoal motility (PSM). Mean TSM was greater (P < 0.05) in SR ejaculates than total ejaculates at 12 and 24 h postejaculation. Mean TSM of undiluted semen was lower (P < 0.05) than other dilution ratios over all periods. Mean TSM was greater (P < 0.05) at a 25 x 10(6)sperm/ml dilution ratio than a 50 x 10(6)sperm/ml dilution ratio at 12 and 24 h postejaculation, and greater (P < 0.05) than a 100 x 10(6)sperm/ml dilution ratio from 3 to 24 h postejaculation. Similar patterns were found for PSM. Collection of SR ejaculates and dilution to 25 x 10(6)sperm/ml improved longevity of spermatozoal motility.     

Germ-Line Transmitted,Chromosomally Integrated HHV-6 and Classical Hodgkin Lymphoma

A unique feature of both human herpesvirus 6A and B (HHV-6A and B) among human herpesviruses is their ability to integrate into chromosomal telomeres. In some individuals integrated viral genomes are present in the germ-line and result in the vertical transmission of HHV-6; however, little is known about the disease associations of germ-line transmitted, chromosomally integrated HHV-6 (ciHHV-6). Recent publications suggest that HHV-6 is associated with classical Hodgkin lymphoma (cHL). Here we examine the prevalence of ciHHV-6 in 936 cases of cHL and 563 controls by screening with a duplex TaqMan assay and confirming with droplet digital PCR. ciHHV-6 was detected in 10/563 (1.8%) controls and in all but one individual the virus was HHV-6B. Amongst cases 16/936 (1.7%) harboured ciHHV-6, thus demonstrating no association between ciHHV-6 and risk of cHL.     

Effect of ejaculation frequency and season on donkey jack semen

Semen characteristics of first and second successive ejaculates from 6 jacks were evaluated weekly for 12 mo. The semen was collected at 4-h intervals, using an artificial vagina with a female in either natural or induced estrus. The statistical analysis was done by factorial delineation 2 x 2 in randomized blocks. Due to some ejaculation failures, the data had to be divided into 2 Groups (A and B) for statistical analysis: Group A - ejaculates preceded by 2 ejaculates in the previous week and Group B - ejaculates preceded by only 1 ejaculate in the previous week. If no statistical difference was observed between the groups in a given parameter, the data was grouped together. Semen characteristics for the first and second ejaculates, respectively, showed the following mean +/- SEM: gel-free semen volume 29.2 +/- 2.2 and 31.7 +/- 2.2 ml; progressive motility 71.0 +/- 1.6 and 72.9 +/- 1.6%; sperm vigor 3.8 +/- 0.1 and 4.1 +/- 0.1; live spermatozoa for Group A 82.6 +/- 2.1 and 82.3 +/- 2.1%, and for Group B 84.6 +/- 1.4 and 86.6 +/- 1.4%; total number of spermatozoa for Group A 10.6 +/- 0.8 x 10(9) and 5.8 +/- 0.8 x 10(9), and for Group B 13.3 +/- 1.2 x 10(9) and 9.6 +/- 1.2 x 10(9); head abnormalities for Group A 1.2 +/- 0.3 and 1.4 +/- 0.3%, and for Group B 1.6 +/- 0.3 and 1.9 +/- 0.3%; mid piece abnormalities 7.7 +/- 0.7 and 6.1 +/- 0.7%; tail abnormalities 7.3 +/- 0.7 and 6.8 +/- 0.7%; pH 7.6 +/- 0.0 and 7.6 +/- 0.0. Significant differences (P < 0.05) were observed between the animals for all sperm characteristics except for sperm vigor. The means for the first and second ejaculates were significantly different (P < 0.05) for the total number of spermatozoa in all the animals, while the percentage of mid piece abnormalities was significantly different in only 1 jack. Seasonal effects on sperm parameters were observed only for semen pH.     

Manual semen collection from a Grevy's zebra stallion (Equus grevyi), onset of sperm production, semen characteristics, and cryopreservation of semen, with a comparison to the sperm production from a Grant's Zebra stallion (Equus burchelli boehmi)

A manual technique was used to collect representative ejaculates from an unrestrained Grevy's zebra stallion beginning at 13 mo of age to determine the onset of sperm production, to calculate the number of spermatozoa produced per ejaculate, and to determine any seasonality associated with sperm production. Spermatozoa first appeared in the ejaculate at 31 mo of age. By 48 mo of age the zebra was producing up to 40 billion spermatozoa per ejaculate. Progressive sperm motility ranged from 75 to 95%. Gel-free semen volume averaged 75 to 120 ml/ejaculate. Gel volume ranged from 0 to 1100 ml/ejaculate. Semen was frozen in 2 different extenders in 0.5-ml PVC straws. The post-thaw motility of cryopreserved spermatozoa ranged from 30 to 70%. A domestic horse mare became pregnant on the first cycle after insemination with frozen-thawed spermatozoa from this zebra. Sperm production data obtained from semen collections made on a Grant's Zebra stallion from 3 to 8 yr of age is presented for comparison of the 2 species.