Electroejaculation Increases Low Molecular Weight Proteins in Seminal Plasma Modifying Sperm Quality in Corriedale Rams

This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and 2‐D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation.     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.     

Effect of method of seminal collection on the retrograde flow of spermatozoa into the urinary bladder of rams

The effects of method of seminal collection and a diuretic on retrograde flow of spermatozoa into the urinary bladder of rams were examined. In experiment 1, semen and urine were collected from 8 rams during the non-breeding season. Prior to seminal collection, all rams were given furosemide and a sample of urine was obtained during micturition. Semen was then collected from each ram with an artificial vagina or by electroejaculation in alternate weeks for 4 weeks, and the urine released during the first postseminal collection micturition was collected in 4 consecutive samples. The volume of electroejaculates was larger (P less than 0.0001) than the volume of ejaculates, but the total number of spermatozoa in the electroejaculate or in the ejaculate were not different (P greater than 0.1). Urine obtained before seminal collection was azoospermic or contained few, nonmotile spermatozoa (mean +/- SD = 0.053 +/- 0.114 x 10(6)/ml). The adjusted spermatozoal concentration (mean +/- SD = 1.630 +/- 2.258 x 10(6)/ml) in the urine collected after seminal collection was 31 times higher (P less than 0.0001) and there were motile spermatozoa in most (97%) of the samples. The spermatozoal concentration in sequential samples of urine was not different (P greater than 0.1) between samples and was not affected (P greater than 0.1) by the method of seminal collection. There was a trend, approaching significance (P = 0.052), for an effect of method of seminal collection on the percentage of retrograde flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of semen collected by pharmacologically induced ejaculation from a stallion with seminal vesiculitis

The present study compared the quality of sperm collected by artificial vagina or pharmacologically induced ejaculation from a 10-year-old thoroughbred stallion with seminal vesiculitis. The pharmacological protocol involved intravenous administration of detomidine (0.01 mg/kg) and oxytocin (20 IU) and successfully induced ejaculation in all attempts of semen collection. Sperm motility, plasma membrane and acrosome integrity (PMAI), reactive oxygen species (ROS) levels, polymorphonuclear neutrophil (PMN) percentage, and bacterial profiles of fresh and cooled semen (5°C for 24 hr) were evaluated. Semen obtained by the pharmacological method presented reduced seminal volume, decreased PMN percentage and superior sperm motility in cooled samples. Moreover, higher PMAI and lower ROS levels were observed in semen collected by the pharmacological method. Therefore, pharmacologically induced ejaculation is an alternative to obtain semen with minimal contamination and with sperm of superior quality and longevity from stallions with seminal vesiculitis.     

A comparison of electro-ejaculation with the artificial vagina for ram semen collection

Nine fertile three-year-old Romney rams were ejaculated both by means of electro-ejaculation and by the artificial vagina. The ram repeatabilities of semen volume, concentration, total sperm number, percentage normal sperm and wave motion score of ejaculates collected by electro-ejaculation were 0.17, 0.08, 0.12, 0.61 and 0.23 respectively compared with the repeatabilities of ejaculates collected by the artificial vagina of 0.28,0.79, 0.56,0.72 and 0.54. The repeatability results showed that it was possible to rank rams on the quantity of sperm produced in a single ejaculate obtained by an artificial vagina, but not on the quantity obtained by electro-ejaculation. The correlation co-efficients between the ram means for the two techniques were for volume 0.05, concentration 0.06, total sperm number -0.21, percentage normal sperm 0.73, and wave motion score 0.35. Only the percentage normal sperm coefficient was significant.     

Seminal Plasma Composition from Ejaculates Collected by Artificial Vagina and Electroejaculation in Guirra Ram

This study was conducted to evaluate changes in ram seminal plasma composition from ejaculates obtained using artificial vagina (AV) and electroejaculation (EE). To address this question, we assessed the effect of semen collection method on volume, sperm concentration, sodium concentration, potassium concentration, sodium/potassium ratio, total protein content and protein profile using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide gel electrophoresis. The main findings from this study were: (i) similar volume was obtained, while sperm concentration was significantly lower for EE method; (ii) potassium and sodium/potassium concentration ratio were not influenced by recovery method, while sodium concentration increased significantly when semen was recovered using EE; (iii) approximately 80% of the total relative seminal plasma protein is represented by four protein fractions of molecular weights around 15, 21, 24 and 50 kDa and there were not differences and (iv) focussing the two-dimensional SDS-PAGE gel on the 10–25 kDa rank, the image analysis software detected around 22 spots with isoelectric points ranging from 5.1 to 6.1. Two protein spots (15 kDa and 5.5 and 22 kDa and 5.2 for molecular weight and isoelectric point respectively) increased significantly when semen was recovered using EE. One spot protein with molecular weight around 25 kDa and isoelectric point of 5.2 were only found in the seminal plasma from the semen recovery by AV. As it was demonstrated, ejaculates obtained with EE modify the sodium concentration, alter two proteins concentration and induced the loss of one protein in seminal plasma.     

CASA Assessment of Kinematic Parameters of Ram Spermatozoa and their Relationship to Migration Efficiency in Ruminant Cervical Mucus

Sperm motility is an indicator of male fertility because of its importance for sperm migration through the female genital tract and for gamete interaction at fertilization. This study analyses the relationship between computer assisted semen analysis (CASA) motility patterns and sperm migration of rams in ruminant cervical mucus. In experiment 1, spermatozoa extended with sperm analysis medium (SAM) and seminal plasma were compared in terms of motility. In experiment 2, 56 semen samples were collected either with artificial vagina (AV) or electroejaculator to be compared in terms of motility performance. In experiment 3, 104 ejaculates collected by AV from 26 males were analysed via the CASA system to characterize their motility patterns. In experiment 4, ejaculates from pairs of rams (20 rams in total) were simultaneously assessed for mucus migration (ovine, caprine, bovine) and motility patterns to evaluate the correlations between both parameters. Semen collected by AV and extended in SAM allows the most reliable assessment for sperm motility. Ram spermatozoa move fast and follow a linear trajectory compared with other ruminants. Continuous line velocity (VCL) and average path velocity (VAP) are the only sperm kinematic parameters that presented significant positive correlations with the ability to migrate in sheep cervical mucus (p < 0.05). Continuous line velocity, VAP, straight line velocity and linearity are highly significantly related with migration efficiency in goat cervical mucus (p < 0.01) and only lateral head displacement is negatively related to sperm migration in bovine cervical mucus (p < 0.05). These results suggest that specific kinematic parameters confer the ability of spermatozoa to colonize and migrate through epithelial mucus with different rheological properties.     

A comparison of quality parameters of fresh feline ejaculates collected by three different collection techniques

The aim of our study was to compare the quality parameters of fresh feline ejaculates collected by three different techniques—urethral catheterization after medetomidine administration (CT), electroejaculation (EE) and epididymal slicing after orchiectomy (EP). A total of 34 adult male cats (Felis catus) were included in the study. In all male cats, the sperm collection was performed under general anaesthesia by three collection methods in the following order: urethral catheterization, electroejaculation and epididymal slicing. The sperm parameters evaluated were as follows: volume, motility, viability, sperm concentration, total sperm count and morphological examination. The highest quality semen parameters were achieved using EE. The comparison of results of the evaluated sperm quality parameters from EE and EP showed significant differences only in one case—the percentage of head abnormalities and lower percentage of head abnormalities were achieved using EE compared to EP: 8.5% (3.0%–21.0%) versus 10.0% (4.0%–22.0%). Semen collected by CT rendered the lowest quality samples when compared to sperm samples collected by EE and EP, especially with respect to the motility and total sperm count which were significantly lower (p < 0.001). Our study showed that sperm samples collected by EE and EP result in better quality of feline ejaculates compared to collection by CT from sperm samples collected from the same male cats. These results demonstrate the necessity of further research of urethral catheterization as a novel technique of semen collection in male cats.     

Ultrasonography of spontaneous lesions of the genital system of three rams, and their influence on semen quality

Three young Suffolk ram lambs had lesions which rendered them reproductively unsound. One had a unilateral scrotal hernia, detected by palpation, and two had sperm granulomas which were detected by trans-scrotal ultrasonography at 18 to 20 weeks of age, before they could be detected by palpation. Changes in the lesions were monitored ultrasonographically at two-week intervals with a 7.5 MHz linear array transducer. On the day of each examination, semen was collected from the rams with sperm granulomas by using an artificial vagina, and their libido was assessed. Semen could rarely be collected from the third ram. Comparisons were made with similar data obtained from two normal rams. When the rams were 20 to 21 months of age, they were vasectomised on the normal side, so that the influence of the lesions on semen composition and libido could be assessed, and semen was collected and evaluated until they were euthanised at approximately 24 months of age, when the genital system was examined for gross lesions. The sperm granulomas changed in size (one of them increased in size by about 30 times) and echotexture over the 19-month period of the study, whereas the scrotal hernia, which contained omental fat, remained fairly constant. The libido of the two rams with sperm granulomas was comparable with the two normal controls. The total mean (sem) number of sperm in the ejaculates were 1.11 (0.10) x 10(9) and 2.03 (0.17) x 10(9) sperm/ml in the affected rams and 4.11 x 10(9) sperm/ml in the normal rams over the same period; the differences were significant (P<0.05). After unilateral vasectomy, the ejaculates from these two rams contained small numbers of dead spermatozoa, mostly with degenerate heads. Postmortem examination confirmed the gross lesions identified ultrasonographically.     

Influence of vitamin E and vitamin E-selenium combination on arginase activity,nitric oxide level and some spermatological properties in ram semen

The effects of vitamin E and vitamin E-selenium combination on seminal plasma arginase activity and nitric oxide level and some spermatological properties in rams were investigated in this study. For control group, animals were injected intramuscularly with physiological saline. For vitamin E group, rams were injected intramuscularly with 300 mg/ram vitamin E. For vitamin E + selenium group, animals were injected intramuscularly with 5 ml/ram vitamin E + selenium. The semen was collected by artificial vagina at 1st, 4th, 24th, 48th and 72nd hr after administration in each group. Significant decreases in seminal plasma arginase activity (at 1st, 24th and 48th hr), nitric oxide level (at 72nd hr) and abnormal sperm rate (at 1st, 24th and 72nd hr), and significant increases in semen volume (at 24th hr), semen mass activity (at 24th and 48th hr), sperm motility (at 24th, 48th and 72nd hr) and concentration (at 1st hr) were observed in vitamin E group compared with control group. Similarly, significant increase in semen volume (at 1st, 24th and 48th hr), mass activity, (at 48th hr), motility (at 48th and 72nd hr) and concentration (at 4th, 24th and 48th hr), and significant decrements in abnormal sperm rate (at 1st, 24th, 48th and 72nd hr), seminal plasma nitric oxide level (at 1st, 4th, 24th and 48th hr) and semen pH (at 24th and 48th hr) were detected in vitamin E + selenium group in comparison to the control group. As a result, it is suggested that vitamin E and/or vitamin E + selenium applications may improve reproductive performance.     

Seminal plasma insulin-like growth factor I and total protein concentration in peripubertal period of the Gyr bulls

This study aimed to evaluate the correlation of seminal plasma IGF-I and total proteins from peripubertal bulls with semen characteristics and onset of puberty. Sixteen dairy Gyr bulls were separated into early (n = 8) and regular (n = 8) groups, according to the onset of puberty. Semen was monthly collected by electroejaculation from 14 to 26 months of age, and the onset of puberty was retrospectively determined (17.0 ± 1.6 and 19.2 ± 1.5 months, to early and regular group, respectively). Five time points were evaluated (day −60, day −30, day 0, day 30 and day 60 days of puberty), being day 0 considered as beginning of puberty. Scrotal circumference and body weight were also assessed. Semen characteristics were evaluated, the seminal plasma was separated by centrifugation and total protein and IGF-I concentrations were determined. There was no difference between groups to concentration of the seminal plasma total proteins, but we found an interaction between group and age. Seminal plasma IGF-I concentrations were not different between early and regular groups; thus, the data were combined for analysis. Combined IGF-I concentrations were positively correlated with sperm motility and concentration, age, body weight and scrotal circumference. Negative correlation was found between IGF-I concentration and total sperm defects. Increased IGF-I was observed in day −30 and day 0 of puberty in early and regular groups, respectively. Seminal plasma total proteins are involved in peripubertal modifications and IGF-I from Zebus dairy bulls can influence the seminal characteristics and the growth factor increase occurs concomitantly with body growth but cannot be used to define puberty bulls earlier.     

Antibiotics for the preservation of koala (Phascolarctos cinereus) semen

Aim To determine the normal microbial flora of the koala ejaculate and prepuce in order to select appropriate antibiotics for addition into diluents designed for the preservation of semen.
Procedure Bacteriological samples of the koala prepuce (n = 12) and ejaculate (n = 20) were submitted for microbial culture and sensitivity testing. Microbial flora of ejaculates collected by electroejaculation and artificial vagina were compared. The effects of varying concentrations of penicillin G and gentamicin on sperm motility and on the growth of bacteria in diluted semen stored at room temperature and 16°C over a 24 h period were investigated.
Results A range of bacteria was isolated from the koala prepuce and ejaculate. The predominant organisms in semen collected by electroejaculation and artificial vagina were Corynebacterium spp, none of which could be assigned to any recognised species. The addition of penicillin G and gentamicin to a PBS-based diluent at dose rates of 1000 to 2000 IU/mL and 100 to 200 m g/mL respectively, resulted in no adverse effect on sperm motility over a 24 h incubation period. Penicillin G (1000 IU/mL) and gentamicin (100 m g/mL) prevented growth of bacterial contaminants in diluted koala semen.
Conclusion By controlling the growth of bacteria in extended koala semen, penicillin G and gentamicin are likely to lengthen the period by which spermatozoa can be stored at 16°C and reduce the possibility of disease transmission during artificial insemination procedures.     

Effect of male accessory gland secretions on sensitivity of porcine sperm acrosomes to cold shock, initiation of motility and loss of cytoplasmic droplets

Hyaluronidase release was used as an index of acrosomal membrane damage during cold shock of epididymal boar sperm and ejaculated sperm from intact and vesiculectomized boars. Sperm were also incubated with seminal plasma from intact and vasectomized boars to examine the contributions of male accessory gland secretions. Acrosomal membranes of epididymal sperm were more resistant to cold shock than those of ejaculated sperm. Only 36% of the hyaluronidase released by ejaculated sperm was released by the epididymal sperm in spite of similar hyaluronidase content of the sperm. Preincubation of epididymal sperm in seminal plasma from both intact and vasectomized boars increased resistance to cold shock by 60 to 80%. Initial dilution of epididymal sperm with seminal plasma, rather than Ringer-fructose buffer, was associated with low progressive motility and with retention of cytoplasmic droplets. In contrast, acrosomal membranes of ejaculated sperm from intact and vesiculectomized boars exhibited similar sensitivity to cold shock, releasing hyaluronidase capable of forming .20 and .19 mumol N-acetylglucosamine from hyaluronic acid/10(8) sperm in 8 min. Moreover, seminal plasma from vasectomized boars had no effect on acrosomal sensitivity to cold shock of ejaculated sperm from vesiculectomized boars.     

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

Lipid and protein oxidation levels in spermatozoa and seminal plasma of Asian Elephants (Elephas maximus) and their relationship with semen parameters

Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid‐reactive species (TBARS) assay and the 2, 4‐dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p < .05). The MDA and PC levels in seminal plasma were negatively correlated with the percentages of progressive motility (p < .05). In addition, the negative correlation between sperm concentration and seminal plasma MDA level was investigated (p < .05). The sperm viability was also negatively correlated with sperm PC level (p < .05). This study indicated that lipid and protein oxidation has deleterious effect on semen quality of Asian elephants.     

Cryodamage of plasma membrane and acrosome region in chicken sperm

Sperm plasma membrane is an essential structure of sperm resistance to freezing. Signs of cryodamage can be visible on the sperm plasma membrane. The aim of our study was to evaluate the appearance of plasma membrane and acrosome in fresh and frozen‐thawed chicken sperm using electron and fluorescence microscopy. Semen was collected from 12 sexually mature roosters of Ross PM3 heavy line, diluted with Kobidil⁺ extender with 16% of ethylene glycol (KEG; control) or with KEG in combination with one of following non‐permeating cryoprotectants: trehalose (KEG‐TRE) or glycine (KEG‐GLY). Fluorescence staining was used for detection of the membrane integrity, apoptotic changes and viability (Annexin V, Yo‐PRO‐1, PI, respectively). Ultrathin sections (70 nm) from samples were prepared to examine sperm head ultrastructure. Freezing process significantly worsened the status of the sperm plasma membranes. In all frozen groups, only about a quarter of the evaluated sperm were graded as class I quality. In the KEG and KEG‐GLY groups, about half of sperm had severe plasma membrane damages (III class). In sperm with extensively damaged membranes (III class), the acrosome–sperm head junction was mostly disturbed. The use of trehalose was more beneficial (p < 0.05) for sperm plasma membrane than the use of glycine. In contrast, a decrease (p < 0.05) in the apoptotic sperm ratio (Yo‐PRO‐1) was noted in the KEG‐GLY group when compared to other treatments. In conclusion, we identified different plasma membrane and acrosome damages in cryopreserved chicken sperm. The loss of acrosomes can contribute to diminishing of fertilization ability of cryopreserved chicken sperm.     

Effect of Post‐Thaw Addition of Seminal Plasma on Motility,Viability and Chromatin Integrity of Cryopreserved Donkey Jack (Equus asinus) Spermatozoa

Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen‐thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post‐thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP‐αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post‐thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post‐thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time.     

A comparison of electroejaculation and epididymal sperm collection techniques in stallions

The purpose of this study was to evaluate 2 methods of semen collection that could be used as terminal procedures in stallions with irreparable conditions, such as fractures or colic. Electroejaculation was attempted under general anesthesia. Forty-eight hours later, the ponies were castrated and 2 different epididymal sperm collection techniques were attempted by using a flushing or floating method. Additionally, the effect of supplemental seminal plasma was evaluated. Experimentally, electroejaculation was found to be a safe but ineffective method of terminal semen collection. Viable sperm cells were successfully recovered with both types of epididymal collection. The flotation method was least cumbersome and showed a tendency to be superior to flushing in terms of sperm motility and percentage of cells passing through glass wool/sephadex filtration, although differences did not reach significance. The addition of seminal plasma to epididymal spermatozoa prior to cryopreservation was of no value. In conclusion, either method of epididymal sperm collection is an acceptable method of terminal semen collection.     

Sperm viability in ram semen diluted and stored in three different extenders.

Semen was collected with an artificial vagina from 4 one-year-old rams, in order to study the changes in sperm motility and membrane integrity of spermatozoa split-diluted and stored at 5 degrees C during 7 days in sodium citrate, Tris, and milk-based extenders, respectively. Sperm motility was assessed subjectively and sperm membrane integrity was determined using the fluorescent probes Calcein-AM and Ethidium homodimer. Representative samples were studied using scanning electron microscopy (SEM). The average incidence of sperm motility decreased over time in all the extenders (p < 0.001). The incidence of spermatozoa showing progressive motility and intact plasma membrane was significantly higher in semen diluted with sodium citrate than in the other 2 extenders following 4 days of dilution until the end of the study. Evaluation with SEM confirmed the findings obtained with the supra vital fluorescent dyes. The results of the present study indicated that there were no differences between sodium citrate-, Tris- or milk-based extenders when ovine liquid semen was stored at 5 degrees C during a short period (2 days). However, when semen was stored for longer time, spermatozoa in the sodium citrate-based extender sustained its viability better.     

Numbers of Sertoli cells, quantitative rates of sperm production, and the efficiency of spermatogenesis in relation to the daily sperm output and seminal quality of young beef bulls

Data from 34 yearling Hereford or Angus bulls were used to investigate relationships of testicular size, quantitative rates of sperm production, Sertoli cell numbers, numbers of germ cells supported per Sertoli cell, and the efficiency of spermatogenesis to daily sperm output and seminal quality. Two ejaculates were collected by electroejaculation from each bull on each of 2 days/week throughout the study. The percentage of progressively motile sperm and the percentage of morphologically normal sperm were determined from aliquots of fresh semen. Additional aliquots of semen were frozen in glass ampules or plastic straws and subsequently evaluated for postthaw motility and percentage of sperm with intact acrosomes. Sertoli cell numbers, the numbers of germ-cells per Sertoli cell, and the efficiency of spermatogenesis were unrelated to the quality of fresh or frozen semen (P greater than 0.05). In first ejaculates, the numbers of sperm and motile sperm were related (P less than 0.05) to testicular parenchymal weight (r = 0.38 and 0.50), daily sperm production (r = 0.45 and 0.53), and spermatids per gram of testicular parenchyma (r = 0.35 and 0.34). Testicular parenchymal weight and daily sperm production also were related to daily sperm output and to the average daily motile sperm output of these bulls (P less than 0.05), but could account for less than 25% of the variability in these end points among bulls.