Seasonal changes of semen quality and freezability in Franches-Montagnes stallions

The objective of this study was to investigate seasonal changes of semen quality parameters in Franches-Montagnes stallions and to compare the freezability of ejaculates collected in autumn and winter. Experiments were performed using 15 stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and evaluated every month during 1 year as well as cryopreserved in autumn and winter (September to February). In fresh semen the gel-free volume, concentration, motility and morphology (normal sperm, major defects, vacuoles and acrosome defects) were evaluated and in frozen-thawed semen the motility as well as the viability (SYBR-14/PI) were performed. To analyse seasonal differences four periods of 3 months each were defined as autumn (September, October, November), winter (December, January, February), spring (March, April, May) and summer (June, July, August). During the 1-year experiment all fresh semen quality parameters demonstrated a clear seasonal and individual pattern. The gel-free volume was significantly (P<0.05) higher in spring and summer compared to autumn and winter while sperm concentration was significantly (P<0.05) lower in spring than at any other time of the year. Total sperm number was significantly (P<0.05) higher and sperm motility significantly (P<0.05) lower in summer than in other seasons. Regarding sperm morphology, normal sperm was significantly (P<0.05) higher in autumn than in winter and summer and major defects were lowest (P<0.05) in autumn. In frozen-thawed semen motility was significantly (P<0.05) improved in the ejaculates collected in autumn compared to winter, while viability showed no obvious differences. Our results clearly demonstrate that individual and seasonal differences occurred in semen quality of Franches-Montagnes stallions. Ejaculates collected in autumn (September, October, November) demonstrated good quality, especially regarding sperm morphology, and were more suitable for cryopreservation because of better motility in frozen-thawed semen collected during autumn than in winter.     

Influence of repeated treadmill exercise on quality and freezability of stallion semen

The objective of this study was to investigate changes of quality and freezability of stallion semen in response to repeated acute treadmill exercise. Ejaculates from 11 stallions were collected, evaluated and frozen weekly during four periods of 4 weeks each defined as before (period 1), during (period 2) and after (periods 3 and 4) intense exercise. In fresh semen the gel-free volume, sperm concentration, motility, normal sperm and sperm with major defects (acrosome defects, nuclear vacuoles, abnormal heads, midpiece defects and proximal droplets) were evaluated. In frozen-thawed semen, motility as well as viability (SYBR-14/PI) were examined. In period 2, all stallions were exercised on an indoor high speed treadmill twice a week (total of eight sessions) using an incremental workload test. Heart rate was monitored telemetrically during exercise and blood samples were taken for determination of cortisol, testosterone and lactate. Results of our investigation demonstrate that heart rate and the plasma concentrations of cortisol, testosterone and lactate significantly (P < 0.05) increased during each exercise session. Furthermore, significantly more major sperm defects were present in periods 3 (69.5+/-2.1%) and 4 (66.8+/-2.1%) than in periods 1 (62.2+/-2.4%) and 2 (62.5+/-2.2%). Acrosome defects increased towards the end of exercise but improved 3 weeks later to values observed before exercise. In frozen-thawed semen, motility was significantly lower in period 2 (45.4+/-2.3%) compared to period 4 (51.6+/-1.7%) and viability was significantly lower in period 2 (49.2+/-2.0%) than in periods 1 (53.8+/-2.1%) and 4 (53.7+/-1.6%). Our results clearly demonstrate that in the stallion repeated strenuous treadmill exercise can negatively influence semen quality and freezability.     

Influence of vedaprofen (Quadrisol) on quality and freezability of stallion semen

The objective of this study was to evaluate the effect of the non-steroidal anti-inflammatory drug (NSAID) vedaprofen (Quadrisol) on quality and freezability of stallion semen. Experiments were performed using 22 Franches Montagnes stallions from the National Stud in Avenches (Switzerland) randomly divided into a control and test group. Vedaprofen was given orally to all stallions of the test group at the recommended therapeutic dose (initial dose of 2mg/kg followed by 1mg/kg body weight every 12h) for 14 days. Control animals received the same amount of carrier substance. During treatment, blood samples of five stallions in both test and control group were collected for PGF(2 alpha)-metabolite (PG-metabolite) determination. Ejaculates from all stallions were collected and cryopreserved weekly for 14 weeks from September to December. Concentrations of PG-metabolite, PGF and PGE were measured in the seminal plasma of ejaculates collected 2 weeks before, during and 2 weeks after treatment. In fresh semen the volume, concentration, motility and number of normal sperm and sperm with major defects (acrosome defects, abnormal heads, nuclear vacuoles, proximal droplets, midpiece defects) were evaluated. In frozen-thawed semen samples motility as well as viability (SYBR-14/PI) were tested and the hypoosmotic swelling test (HOS) was performed. Results demonstrate that vedaprofen had no effect on blood plasma concentration of PG-metabolite but significantly inhibited both, PGF and PGE concentrations in seminal plasma. Furthermore, all quality parameters in fresh and frozen-thawed semen were not affected by vedaprofen treatment but the time of semen collection had a significant (P<0.05) effect on motility, normal sperm and sperm with nuclear vacuoles in fresh semen.     

Influence of Eqvalan (ivermectin) on quality and freezability of stallion semen

The objective of this study was to evaluate the effect of Eqvalan (ivermectin) on stallion semen quality and freezability. Experiments were performed using 22 Freiberger stallions, randomly divided into a control and test group. Semen was collected once a week for 17 weeks from October 1997 to February 1998. Eqvalan was given orally to all stallions of the test group at a therapeutic dose of 0.2 mg ivermectin/kg. Besides measuring the scrotal width, ejaculates were collected to determine the volume, concentration, and the motility and morphology (normal sperm, major defects, vacuoles and acrosome defects). In addition, the motility and viability (fluorescence staining with propidium iodide/SYBR-14) were tested in all frozen-thawed semen samples. During the experimental period, stallions treated with Eqvalan had significantly better concentration (P < 0.0001) and motility (P < 0.0001) in fresh semen compared to control animals. After freezing-thawing, the motility (P < 0.0001) and viability (P = 0.0003) of semen also increased significantly for treated stallions. Regarding morphology, normal sperm (P < 0.0001), major defects (P = 0.0027) and vacuoles (P = 0.0236) were significantly better in the Eqvalan group. In addition to group differences we also observed a time effect on morphological parameters as shown by a decrease of normal sperm and an increase of major defects in both groups during the experiment. Our results demonstrate that a single oral application of Eqvalan did not negatively influence the quality and freezability of stallion semen in the nonbreeding season. Rather, it seems that Eqvalan has a favorable effect on stallion fertility as most sperm parameters examined were significantly improved in treated animals compared to control.     

Seasonal variation in freezability of buffalo semen

Fifty-six buffalo semen ejaculates of mass activity greater than plus three on a five-point scale (28 each in summer and winter seasons) were frozen in Tris yolk glycerol (TY-G) extender with four hours equilibration time. Motility of semen was checked after first extension (initial motility), after equilibration time, 15 minutes after freezing, and 30 days after freezing. Motility increased during winter season. Significant differences (P<0.01) in motility after freezing were observed between summer and winter seasons.     

Seasonal influence on semen traits and freezability from locally adapted Curraleiro bulls

Studies were conducted to characterize the effect of season of the year on testicular morphology, fresh and frozen/thawed semen quality from Curraleiro (Pé-duro) bulls in the Brazilian Central west region. Five adult, healthy bulls underwent an andrological examination and semen collection using an electroejaculator, once a month for a year. Fresh and thawed semen were evaluated for progressive sperm motility and sperm vigor, sperm morphology and acrosomal integrity. Testicular length and volume were less (P<0.05) in April than in the other months of the year. For fresh semen, the ejaculate in April had less volume and sperm concentration (P<0.05), while sperm vigor was less (P<0.05) in June, increasing in January and February. With the frozen/thawed semen, the proportion of sperm was greater (P<0.05) in April to July, decreasing from October to December. Semen collected in December had the greatest (P<0.05) proportion of major defects while that collected in February/March had the highest proportion of minor defects. The proportion of live intact sperm reduced progressively from December to April/May. The marginal influence of the time of the year on testicular biometry and fresh semen in Curraleiro bulls shows the adaptation of this breed to the environmental conditions in the region. Thus, reproduction with natural mating should be successful at any time of year. For frozen semen collection for conservation programs, the best time of year is from June to September.     

Mycoplasmas in stallion semen

Eleven mycoplasma strains were isolated from the semen of 24 stallions. Eight of these strains metabolized glucose and three hydrolyzed arginine. Serological examination by growth inhibition test (GIT) did not allow these strains to be identified. Arginine-degrading strains were not inhibited by antisera against three human mycoplasma strains, M. arginini and M. equirhinis. It was shown, however, by GIT, that all of the glucose-positive strains were antigenically related but that the three arginine-positive strains had a different antigenic structure.A comparison of indices from routine semen examination and certain biochemical components of the semen plasma from ejaculates with and without mycoplasma showed statistically lower levels (P ? 0.01) of glycerylphosphorylcholine, ergothioneine, fructose and total protein in the semen plasma of infected ejaculates than in that of ejaculates without mycoplasma.     

Prostaglandins in stallion semen

The purpose of the experiment was to obtain preparatory information about the presence of prostaglandins in semen collected from various types of horses after different periods of sexual rest. Semen was collected with an artificial vagina. Prostaglandin-like activity was estimated by the bioassay procedure described by Vane (1). Results are expressed in ng/ml PGE(2) of seminal plasma. The total concentration of prostaglandins in the full ejaculate averaged 43.73 +/- 4.93 ng/ml of plasma while the total amount of prostaglandins in the ejaculate was 1076 ng. Taking into consideration the period of sexual rest in the stallion, statistically significant differences were found in the prostaglandin level in the semen of all the stallions.     

Testicular endocrine function, seasonality and semen quality of the stallion

To gain further information on gonadal function of the stallion, concentrations of testicular steroids in blood plasma (bpl) and seminal plasma (spl) and their distribution in the ejaculate were determined. Blood and semen samples from a total of 11 stallions were collected from November to July. Estrone (E1), estrone sulfate (E1S), estradiol-17beta (E2beta) and testosterone (T) were determined in bpl and spl, and in addition androstenedione (A), dehydroepiandrosterone (DHEA) and 5alpha-dihydrotestosterone (5alpha-DHT) were measured in spl. At certain points of time, aliquots of an ejaculate were centrifuged, washed and the distribution of E1, E1S, E2beta and T into seminal plasma and the sperm fraction was assessed. Hormone assay was by RIA, partly after prior separation by HPLC. Mean concentrations (X(g) x DF) were as follows: E2beta (bpl) 31.1 (1.16), (spl) 24.2 (1.42) pg ml(-1); E1 (bpl) 143.3 (1.21), (spl) 117.7 (1.53) pg ml(-1); E1S (bpl) 157.3 (1.44), (spl) 2.92 (1.42) ng ml(-1); T (bpl) 570.6 (1.43), (spl) 23.1 (1.68) pg ml(-1); A (spl) 17.9 (1.39) pg ml(-1); DHEH (spl) 12.4 (1.51) pg ml(-1); 5alpha-DHT (spl) 9.7 (1.29) pg ml(-1). Except for E2beta and A in seminal plasma, a seasonal pattern was established for all other steroids with lowest mean values occurring from November to April. From the semen parameters determined, only motility was correlated to season. There was a higher correlation among oestrogen in blp than in spl and the only correlation identified between oestrogenic and androgenic steroids was between T and E2beta in blp. In spl, T was correlated with A and 5alpha-DHT. T was the dominant free steroid in bpl while it was E1 in spl; T and E1S concentrations were about 23- and 54-fold lower in spl compared to bpl with E1S, however, showing the highest absolute values in both fluids. In the fractionated ejaculate an association of free oestrogens, particularly E2beta, with spermatozoa was observed.     

The effect of x-radiation upon the quality and fertility of stallion semen

The exposure that stallion semen might receive during examination using an airport x-ray security screening system was found to be between 0.5 and 1.0 micro Sieverts (μSv). Ejaculates from 2 stallions were diluted 1:4 (volume:volume) using a nonfat dried milk-glucose extender. A total of 6 ejaculates from each stallion was collected, and each ejaculate was divided into 3 aliquots and these were then exposed to x-radiation at a dose of 0, 1.0, or 10.0 (μSv. Semen quamy was examined immediately post exposure, and the aliquots were then placed into a water bath at 37 °C, after which sample longevity was evaluated.In a second trial, 3 groups of 8 pony mares were inseminated with semen that had been exposed to x-radiation at doses of 0, 1.0, or 10.0 μSv. An entire ejaculate was irradiated and inseminated into each mare on one occasion during estrus, based upon ultrasonographic evaluation of the reproductive tract.After exposure to x-radiation there were no differences among the 3 treatment groups for spermatozoal motility, morphology, or longevity. The 14-d pregnancy rates for the 3 treatment groups were 0 μSv (7 mares), 1.0 μSv (8 mares), and 10.0 μSv (7 mares). One mare (0 (μSv) aborted at 65 d of pregnancy; 21 mares had a pregnancy of normal length, with each delivering a foal at term, although 1 foal died at parturition (1.0 μSv).These findings indicate that the exposure of stallion spermatozoa to x-radiation up to doses of 10 μSv does not have deleterious effects upon spermatozoal motility, morphology, longevity or fertility. The exposure received during examination using an x-ray security screening system is likely to be lower than this dose.     

Developments in stallion semen evaluation

The conventional approach to evaluation of stallion semen dates back several decades, and includes evaluation of spermatozoal concentration, semen volume, spermatozoon morphological characteristics, and spermatozoal motility patterns initially and following in-vitro storage. While an analysis performed in this manner does have predictive value, incorporation of some more newly developed techniques may improve the predictive value of the examination. This communication addresses some newer tests that can be applied today for evaluation of semen, as well some tests that may be available in the coming years.     

Effect of dietary antioxidant supplementation on fresh semen quality in stallion

In this study, the effect of dietary supplementation of organic selenium, vitamin E, and zinc on raw semen characteristics was evaluated. Ten stallions with normal fertility were divided into two groups: a control group (CG), in which standard diet was provided, and a treated group (TG), in which the standard diet was supplemented with 1500 mg of α-tocopherol acetate, 360 mg of zinc, and 2.5 mg of organic selenium on a daily basis. Semen parameters on fresh semen were evaluated three times in all stallions before antioxidant supplementation (T0) and 30 (T1), 60 (T2), and 90 (T3) d after supplementation. Dietary supplementation with experimental antioxidants resulted in a significant increase in average path velocity (121.9 ± 3.1 μm/sec in TG vs 118.9 ± 4.3 μm/sec in CG), straightness (86.2 ± 2.4 % vs 82.6 ± 3.9 % in TG and CG respectively), viability (75.6 ± 10.2 % in TG vs 72.3 ± 6.9 % in CG) and total seminal plasma antioxidants levels (2.7 ± 0.5 mmol/l vs 1.9 ± 0.4 mmol/l in TG and CG respectively) while progressive motility 69.7 ± 11 % vs 62.2 ± 9.3 % in TG and CG stallions respectively) and abnormal sperm morphology (8.2±1.5 % in TG vs 14.4±4 % in CG) significantly improved in treated stallions after 60 d of supplementation. In contrast with previously reported in other species, a negative effect of antioxidant supplementation on semen concentration was recorded in the TG. A positive correlation between progressive motility and total antioxidants in seminal plasma in both treated and control stallions suggested that motility is affected by oxidative-antioxidative status, and that dietary antioxidant supplementation could increase the ability of spermatozoa to contrast reactive oxygen species or the ability of seminal plasma to reduce the oxidative stress. The improvement of semen parameters after antioxidant supplementation was not linear, and after 30 d (or 60 d for some parameters), a further increase was not noted. This evidence suggested that in our standard conditions, dietary intake of these antioxidants could be slightly under the dietary requirement and further evaluation of the actual nutrition requirements of organic selenium, zinc, and vitamin E in the stallion are needed.     

Seasonal changes in semen quality and correlation with plasma hormone profiles in Karan Fries bulls

The aim of the study was to assess the influence of summer and winter seasons on semen quality and plasma hormone concentrations in cross-bred bulls. Semen was collected by an artificial vagina from eight bulls and microscopically evaluated for quality parameters. Semen volume was higher in summer season (p < 0.05) than winter season, whereas nonsignificant variation (p > 0.05) was observed in mass motility, individual motility, sperm viability, sperm concentration and percentage of membrane-intact and acrosome-intact spermatozoa. Plasma prolactin and testosterone concentration were significantly (p < 0.01) higher in summer season than winter season. Plasma testosterone levels were positively correlated with semen volume and negatively correlated with individual motility (p < 0.05). Prolactin showed a significant positive correlation with semen volume. A well-defined seasonal pattern in semen characteristics was not observed and few correlations existed between plasma hormone levels and semen characteristics in Karan Fries bulls.     

Sephadex and sephadex ion-exchange filtration improves the quality and freezability of low-grade buffalo semen ejaculates

The effect of sephadex and sephadex ion-exchange filtration on the improvement in quality and freezability of low-grade buffalo semen ejaculates was assessed. Two types of filtration columns were used: one containing only sephadex G-10 (FS) and the other sephadex G-10 along with ion-exchangers (diethyl amino ethane-52 (DEAE-52) cellulose and carboxy methyl-52 (CM-52) cellulose; FS+IE). Unfiltered samples served as controls. Semen ejaculates extended in Tris-citric acid (1:4) (n=16; initial motility 40-50%) were filtered at the rate of 1.5 ml/min under negative pressure at room temperature (28-30 degrees C). The mean recovery rate (%) of motile spermatozoa in the FS (85.9+/-1.51) and FS+IE (77.10+/-2.28) filtrates did not differ significantly. Percentages of sperm motility, normal acrosomes, and intact plasma membranes were highest (P<0.05) in FS+IE, intermediate (P<0.05) in FS and lowest (P<0.05) in controls at the three stages of cryopreservation (postfiltration final dilution, after equilibration, and after freezing). Mean sperm abnormalities were lowest (P<0.05) in the filtrates of FS+IE, moderate (P<0.05) in FS and highest in controls at all stages of freezing. Compared to dilution and equilibration, freezing greatly reduced (P<0.05) the overall percent motility, normal acrosomes and intact plasma membranes. The spermatozoa eluted through FS+IE columns proved more resistant (P<0.05) in bearing dilution, equilibration, freezing and thawing stresses than the spermatozoa from FS and control samples. It is concluded that filtration systems containing an FS+IE column can effectively enhance the quality and freezability of extended, low quality buffalo semen.     

Bacteriology of preserved stallion semen and antibiotics in semen extenders

Three experiments were conducted to evaluate the effects of different antibiotics in a milk-glucose semen extender on motility of equine sperm and elimination of bacteria following storage of extended semen in vitro. In Experiment 1, 7 antibiotics were compared: amikacin, gentamicin, streptomycin, potassium penicillin, sodium penicillin, ticarcillin, and polymixin B. In Experiment 2, 3 antibiotic treatments were compared: potassium penicillin G, amikacin, or a combination of potassium penicillin G and amikacin. In Experiment 3, 3 antibiotic treatments were compared: potassium penicillin G-amikacin, ceptiofur, and a combination of ticarcillin and clavulanic acid (Timentin). Control treatments (antibiotic-free extender) were included in each experiment. Six motility variables were evaluated: percentage of motile sperm; percentage of progressively-motile sperm; percentage of rapidly-motile sperm; mean curvilinear velocity; mean average path velocity; and mean straight-line velocity. In Experiment 1, mean percentages of motile, progressively motile and rapidly motile sperm were lower (P < 0.05) in semen exposed to polymixin B then in other treatments. Mean average-path velocity of sperm in extender containing polymixin B was lower (P < 0.05) than that of all other treatments, with exception of control or ticarcillin. Mean straight-line velocity of sperm in extender containing polymixin B was lower (P < 0.05) than that of all other treatments, with exception of control, streptomycin or ticarcillin. Semen samples containing gentamicin, amikacin, streptomycin, or potassium penicillin were more effective (P < 0.05) at eliminating bacterial growth than those samples containing polymixin B. Semen samples containing gentamicin were also more effective (P < 0.05) at eliminating bacterial growth than those samples containing ticarcillin or sodium penicillin. In Experiment 2, mean percentage of rapidly-motile sperm, and mean curvilinear, average-path, and straight-line velocities were greater (P < 0.05) for potassium penicillin-amikacin than values for all other treatments. In 2 of 3 stallions, an effect of treatment on percentage of motile sperm was detected (P < 0.05). For one stallion, mean motility of potassium penicillin-amikacin was greater (P < 0.05) than that of all other treatment groups. For another stallion, mean motility of the control was lower (P < 0.05) than that of the other treatments. Following storage, potassium penicillin (16/18 [89%]) or potassium penicillin-amikacin (17/19 [94%]) were more effective (P < 0.05) at controlling aerobic and anaerobic bacterial isolates in semen specimens than was amikacin (10/18 [56%]). In Experiment 3, a difference among treatment groups for motility variables was not detected (P < 0.05). No bacterial growth was recovered in antibiotic-treated semen, with exception of Micrococcus sp. (2 colonies) which were isolated from one semen specimen treated with ceptiofur.     

Oxytocin in the semen and gonads of the stallion

It has been suggested that oxytocin is involved in sperm transport and motility in domestic animals. Immunoreactive oxytocin was measured in seminal fractions (pre-ejaculatory fluid, seminal plasma, gel and sperm) and in extracts of testis and epididymis from stallions. In addition, sections of gonadal tissue from stallions were immunostained for the presence of oxytocin and its neurophysin. Oxytocin was detected in all of the seminal fractions, being highest in the gel. It was also present in washed, lysed sperm and in extracts from the testis and epididymis. Immunostaining for oxytocin was present in occasional interstitial cells in the testis and in the epididymal epithelium and smooth muscle. However, immunostaining for neurophysin was detected in a few interstitial cells in the testis of only 1 of 8 stallions and was absent from all areas of the epididymis. These data demonstrate for the first time the presence of oxytocin in stallion semen and gonadal tissue; however, lack of immunostaining for neurophysin indicated that it was unlikely that there was local synthesis within the gonads.