Seasonal changes in semen quality and freezability in the Warmblood stallion

The objective of this study was to investigate seasonal changes in stallion semen quality and to determine the best time for semen cryopreservation. Experiments were performed using 10 Warmblood stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and frozen every other week during 1 year from January to December 1999. Volume, concentration, and motility, and the number of morphologically normal sperm and sperm with major defects (abnormal heads, acrosome defects, nuclear vacuoles, proximal droplets, abnormal midpieces) were evaluated. For all frozen-thawed semen samples motility as well as viability (SYBR-14/PI) was tested, and the hypoosmotic swelling test (HOS) was performed. To analyze seasonal differences 4 periods of 3 months each were defined: autumn (September, October, November), winter (December, January, February), spring (March, April, May) and summer (June, July, August). During the 1 year experiment all semen quality parameters showed a clear seasonal pattern. The volume, total sperm count and motility in fresh semen were significantly higher (P<0.05) in summer than in winter, while sperm concentration was significantly lower in summer compared to the other seasons. Regarding morphology, normal sperm was significantly lower (P<0.05) in summer than at any other time of the year and higher values (P<0.05) were found for major defects in summer than in spring and autumn. In frozen-thawed semen motility was significantly (P<0.05) improved in autumn when compared to spring and summer. Viability was lowest in summer and differed significantly (P<0.05) from other seasons. The HOS test revealed significantly more (P<0.05) membrane damaged spermatozoa in winter than in spring, summer and autumn. Our results demonstrate that in our climatic conditions clear seasonal differences occur in semen quality of fresh and frozen-thawed semen and that cryopreservation of stallion semen should preferably be performed in autumn.     

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.     

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.     

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008–2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24 h at 17 °C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P < 0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season’s influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.     

Collection method, season and individual variation on seminal characteristics in the llama (Lama glama)

The objective of the present study was to evaluate the effect of semen collection method (electroejaculation "EE" as compared with the artificial vagina "AV"), the season (summer versus winter) and the male used on macroscopic and microscopic characteristics of ejaculates in llamas. A total of 110 ejaculates were collected from six males and 92 of them were analyzed. Ejaculate volume, concentration, total sperm and the following sperm characteristics were studied: motility, membrane function (HOS test), membrane integrity (CFDA/PI fluorochromes) and morphology. A mixed linear model, that considered season and collection method as the fixed variables and the male as the random variable, was used for the statistical analysis. Variability was found between males (p相似文献   

Comparison of semen characteristics,sperm freezability and testosterone concentration between Duroc and Yorkshire boars during seasons

This study was carried out to investigate the effects of Duroc and Yorkshire boars and seasons influencing semen characteristics, frozen-thawed sperm viability and serum testosterone concentration. Yorkshire boars produced higher semen volume compared to Duroc boars among seasons. However, sperm concentration did not differ significantly between Duroc and Yorkshire boars among seasons. Sperm motility of frozen-thawed sperm in Yorkshire boars was higher than in Duroc boars in spring and summer. Normal acrosome of frozen-thawed sperm in Yorkshire boars was higher than in Duroc boars in winter. Serum testosterone concentrations in Duroc and Yorkshire boars were higher in spring than in summer, autumn and winter. Serum testosterone concentrations in spring and summer were higher in Yorkshire boars than in Duroc boars. In conclusion, when serum testosterone concentrations were higher in Duroc and Yorkshire boars among seasons, semen volume, sperm concentration and frozen-thawed sperm viability were higher.     

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.     

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.     

Seasonal functional relevance of sperm characteristics in equine spermatozoa

A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus). Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September-December, January-March, late March-May, and June-July. Parameters monitored included sperm concentration, sperm motility, sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential. Overall, seminal parameters monitored are affected mostly by time period, followed by animal and lastly by fertility, stressing the importance of individual variations in out-bred animal models. The analysis of multiple ejaculates from the same animals showed clear seasonal-based differences (P < 0.05) with poor semen quality in winter and a noticeable improvement in sperm quality with increasing photoperiod. Better semen quality was observed between late March and May. Interactions between month period, animal, and fertility were evident (P < 0.05) for sperm concentration, head and tail sperm anomalies, and acrosomal integrity. Thus, it may be advisable to adjust the use of stallion semen according to seasonal variations.     

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.     

Influences of a diet supplemented with linseed oil and antioxidants on quality of equine semen after cooling and cryopreservation during winter

Seasonal changes in the reproductive physiology of stallions contribute to a decrease in the quality of frozen-thawed semen during late winter. Changes in the lipid composition of the sperm plasma membrane may contribute to this phenomenon. In the present study, we have, therefore, investigated the effects of adding linseed oil (LO) in combination with antioxidants to the diet of breeding stallions on the motility and membrane integrity of cooled–stored and cryopreserved semen. Starting in November, the diet of LO stallions (n = 6) but not control (C) stallions (n = 5) was supplemented with LO (100 mL once daily) plus an antioxidant (Myostem Protect; Audevard, Clichy, France) for a total of 84 days. Before (November) and at the end of this period (February), ejaculates were processed for cryopreservation (n = 3 ejaculates per stallion) and cooled shipping at 5 °C. Frozen-thawed and cooled–shipped semen was sent to the laboratory for computer-assisted semen analysis of total motility, progressive motility, and velocity parameters (average path velocity [VAP], curved line velocity [VCL], and straight-line velocity [VSL]) and evaluation of membrane integrity. The quality of frozen-thawed semen decreased (P < 0.05) from November (e.g., total motility LO 69 ± 3% and C 67 ± 3%) to February (total motility: LO 55 ± 4% and C 59 ± 3%) independent of treatment (P > 0.05). A decrease in the velocity parameters VAP, VCL, and VSL was more pronounced in LO stallions than in C stallions (e.g., VSL: November LO 67 ± 1 μm/s, C 64 ± 2 μm/s; February LO 59 ± 2 μm/s, C 63 ± 2 μm/s; interaction month by treatment, P < 0.05). In cooled–stored semen, total motility, progressive motility, and membrane integrity were lower in February than in November (P < 0.001 for all parameters). Supplementation of the diet with LO and antioxidants attenuated this decrease (e.g., Day 1 of cooled storage = 24 hours after semen collection: total motility in November LO 88 ± 1% and C 87 ± 3%; in February LO 83 ± 2% and C 73 ± 11%; interaction month by treatment: P < 0.05). Velocity parameters VAP, VCL, and VSL were significantly lower in February than in November (P < 0.001), but this decrease was not affected by treatment. In summary, dietary supplementation of stallions with LO plus antioxidants attenuated a decline in motility and membrane integrity of cooled–stored stallion semen during winter. This may improve the fertility of cooled–shipped semen. In contrast, the treatment did not counteract the decrease in quality of frozen-thawed semen that occurs in late winter.     

The seasonal fertility of North American bison (Bison bison) bulls

The objective of this study was to determine the presence and magnitude of seasonal fluctuations in semen quality and other reproductive indices in bison bulls. Testicles from 288 commercially slaughtered bison bulls were collected monthly over a 1-year period. Carcass and testicle weight were determined and measurements of seminiferous tubule lumen, diameter, and epithelial thickness were made. Sperm cell morphology and defects were described and quantified using epididymal semen from each testicle. Twenty-one Plains (Bison bison bison) and Wood bison (Bison bison athabascae) breeding bulls, averaging 6.0 years of age (range 2.5-8.0), from three farms were selected for semen collection and evaluation on the basis of producer co-operation. Semen was collected by electro-ejaculation on four seasonal occasions during a 12-month period. Ejaculate quality was judged on the basis of volume, density, gross and individual motility, morphology, live/dead ratio, and concentration. Sperm cell morphologies were evaluated microscopically and classified according to criteria used for bovine semen. Fecal testosterone was measured at each semen collection using a commercial competitive binding radioimmunoassay. There was an increase in carcass weights over the study period and testis weights were moderately correlated (r=0.44) with carcass weights. However, mean testes weights were heavier (P<0.05) in the summer than in winter, spring, or fall periods. There were no differences in the proportion of normal and abnormal epididymal sperm between seasons but there were seasonal changes in the testicular parenchyma. Seminiferous tubule and lumen diameter, and epithelial thickness were greatest (P<0.05) in summer. Live bulls gained weight between April and November, but lost weight over the winter. Normal sperm cell percentages as well as individual sperm cell motility in electro-ejaculated sperm samples were higher (P<0.05) at the pre-breeding collection relative to other collections, but no change in sperm cell concentrations occurred over the study period. Fecal testosterone concentrations were highest at the pre-breeding period (June) but decreased (P<0.05) in each subsequent collection to reach their lowest levels in the April. While many changes in seen characteristics were not significant, overall results indicate the presence of some reproductive seasonality and increased testicular capacity in the summer breeding season. Bulls showing marginal semen quality in the winter but otherwise carrying desirable genetic traits may warrant another evaluation in late spring prior to being culled from a breeding program.     

Environmental factors contributed to circannual rhythm of semen quality

We investigated whether human semen parameters present circannual rhythm or not, and whether environmental factors exert on semen quality. This retrospective study used data of patients mainly from Reproductive Medicine Center and Urology and Andrology Clinic of a general hospital in China. Sperm concentration and motility were measured by computer aided sperm analysis (CASA). Sperm morphology was scored based on the strict criteria (WHO, 2010). The Kruskal–Wallis rank test was used to investigate the relationship between semen parameters and season/month. Partial correlation coefficients were used to analyze the relationship between semen parameters and environmental factors. In this study, we found that sperm concentration and total amount per ejaculate were significantly lower in summer and higher in winter. But, sperm progressive motility and motility were significantly higher in spring and summer (from March to June), lower in autumn and winter (September and October). Unexpectedly, normal sperm morphology and mixed agglutination reaction (MAR) positive rate didn’t vary along with season or month. Furthermore, temperature was negatively related to sperm concentration and total amount per ejaculate. Precipitation was positively associated with progressive motility and normal sperm morphology, but negatively related to sperm head defect percentage. The length of sunlight was positively related to progressive motility. The Air Quality Index (AQI) was positively associated with semen volume and sperm total amount per ejaculate. These suggest seasonal and monthly variation underlying some semen parameters.     

Characteristics and seasonal variation in the semen of Markhoz bucks in western Iran

The aims of the present study were to evaluate if seasonality in semen characteristics and plasma testosterone concentrations exist in Markhoz male goats. Ten Markhoz (Angora) bucks were housed and fed according to standard recognized practices. During the observation period, semen was collected monthly with the aid of an electro-ejaculator and examined microscopically immediately after collection. Physical parameters of semen and the semen index were recorded. Blood samples were also taken monthly throughout the observation period and the plasma testosterone concentration monitored. Bucks demonstrated a higher semen quality (P < 0.05) in autumn and summer (semen index of 965 × 10⁶ and 752 × 10⁶ ml⁻¹, respectively), compared to spring and winter (semen index of 606 × 10⁶ and 512 × 10⁶, respectively). This coincided with a higher (P < 0.05) plasma testosterone concentration in autumn and summer (8.1 and 10.1 ng ml⁻¹, respectively), compared to that obtained in spring (3.0 ng ml⁻¹) and winter (2.5 ng ml⁻¹). During autumn and summer, the ejaculate volume (average of 1.2 and 1.0 ml), sperm output (1159 × 10⁶ and 1005 × 10⁶ sperm ml⁻¹), sperm mass motility (4.2 and 4.3), sperm progressive motility (83.9 and 82.0%) and percentage live sperm (90.7 and 88.2%, respectively) of the bucks were higher (P < 0.05) than in the spring (0.6 ml, 880 × 10⁶ sperm ml⁻¹, 3.3, 71.5% and 80.2%) and winter (0.7 ml, 863 × 10⁶ sperm ml⁻¹, 4.0, 71.5% and 84.9%, respectively). During autumn and summer, the percentage of sperm abnormalities (5.0 and 9.2%) was significantly lower than that in spring (12.9%) and winter (11.2%). The semen pH was slightly alkaline being significantly (P < 0.05) lower in the autumn (7.1) than in spring (7.3). Data showed season of the year to influence all semen parameters evaluated—indicating that optimal buck performance may be obtained in late summer and autumn. It can thus be said that Markhoz bucks have distinct seasonal spermatogenic activity, with poorer semen characteristics being recorded during winter and spring. This may be a critical obstacle when implementing an intensive breeding system of three kidding seasons in 2 years, with natural mating being implemented.     

Motion characteristics of Murrah buffalo bull spermatozoa in various seasons and its relationship with functional integrity of the plasmallema

Semen was collected from six adult (3.5-7-year-old) Murrah buffalo bulls at weekly intervals for 1 year and evaluated for routine parameters, motion characteristics, reactivity in hypoosmotic solution, and acrosomal and other morphological abnormalities of the spermatozoa. The overall motility (MOT), straight line velocity (VSL), curvilinear velocity (VCL), linearity (LIN), lateral head displacement (ALH) and average path velocity (VAP) were 66.85+/-2.79%, 26.58+/-0.24 and 107.07+/-1.47 microm/s, 26.91+/-0.01%, 11.19+/-0.09 and 61.78+/-2.79 microm/s, respectively. Significant seasonal variation was observed in sperm kinematics and hypoosmotic swelling (HOS) reactivity. Except for LIN, the mean values of sperm dynamics were higher during summer and rainy season and significantly lower in winter season. Sperm kinematics showed significant (P<0.01) positive correlation (r=0.25-0.60) with plasmallemal integrity. Ejaculates with less than 50% HOS-reactive spermatozoa had significantly lowered MOT, VSL, VCL and VAP as compared to the ejaculates with >50% HOS-positive spermatozoa. No significant difference was observed in sperm kinematics among the ejaculates having 50-70% and >70% HOS-reactive spermatozoa. The trend of motion dynamics of the spermatozoa with respect to HOS reactivity was similar in all the three seasons (summer, rainy and winter). The results indicate that ejaculates having more than 50% of HOS-reactive sperm show a higher magnitude of sperm kinematics compared to ejaculates having less than 50% HOS-positive spermatozoa.     

Seasonal variations in the composition of ram seminal plasma and its effect on frozen-thawed ram sperm

It has been proposed that seminal plasma (SP) in the extender or in post-thaw media can prevent and revert cold-shock damage in cryopreserved ram sperm; however, this was dependent on season. We evaluated sperm parameters from Frisian ram semen incubated for various intervals with SP from all seasons and stored at -18 or -196 degrees C. At both temperatures, SP from autumn or winter increased (P<0.05) sperm motility, whereas no SP, or SP from spring or summer, had no effect. However, neither viability nor membrane or acrosomal status were modified by SP. Thirteen SP proteins were bound to the sperm surface (16.1, 16.7, 17.4, 23.3, 25.2, 27.5, 35.0, 40.0, 49.0, 53.5, 55.5, 61.0, and 86.0kDa). The SP proteins that bound to sperm were affected by season, but not by conservation temperature. Sperm incubated with SP from autumn had increased concentrations of five proteins; two were identified (with specific antibodies) as RSVP14 and RSVP20. In conclusion, SP from autumn and winter improved sperm motility of frozen-thawed ram sperm, and storage of ram SP at -18 or -196 degrees C did not affect protein composition. The SP proteins that bound to the sperm surface may be responsible for sperm membrane stabilization and should be further investigated.     

Quality of stallion semen obtained by a new semen collection phantom (Equidame) versus a Missouri artificial vagina

A study was performed to test a new semen collection device (Equidame phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame phantom with that obtained by a Missouri artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw and chilled semen samples were cultured and the number of bacterial colonies counted after incubations of 24 and 48 h. After a 24-h incubation the number of colony-forming units (CFU) in raw semen was significantly higher (P<0.01) when collected by the Missouri artificial vagina than by the Equidame phantom. After cooled storage, 75% of the semen samples contained no bacteria after an incubation of 24 h, and 69% yielded no growth after 48 h. The sperm-rich fractions (Cup 2) collected by the Equidame phantom had lower mean volumes (22.1 +/- 2.3 mL [+/- SEM] versus 101.6 +/- 9.3 mL) and significantly higher mean sperm concentrations (218.0 +/- 25.8 x 10(6) vs 86.2 +/- 8.1 x 10(6) cells/mL; P<0.05) than the total ejaculates collected by the Missouri device. The total and progressive motility of chilled and frozen-thawed semen did not differ significantly between collection methods. The Equidame phantom yielded semen that was of a lower bacteriological colony counts, but had sperm motility similar to that of semen collected with the traditional method by the Missouri artificial vagina.     

Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation

Effects of seminal plasma on post-thaw motility and membrane integrity of cryopreserved horse spermatozoa were investigated. Carboxyfluorescein diacetate staining was used for the assessment of sperm membrane integrity. Adding 30% of seminal plasma from stallions with high post-thaw sperm motility to ejaculates from stallions with low post-thaw sperm motility increased progressive motility from 24.0 +/- 1.6 to 34.5 +/- 1.9% (P < 0.05) and membrane integrity from 27.0 +/- 2.1 to 34.3 +/- 2.3% membrane-intact spermatozoa (P < 0.05). Conversely, the addition of seminal plasma from stallions with low post-thaw sperm motility to ejaculates from stallions with high post-thaw motility decreased progressive motility from 36.0 +/- 1.6 to 30.0 +/- 2.7% (P < 0.05) but did not induce changes in membrane integrity. Seminal plasma from stallions with opposite post-thaw motility therefore clearly influenced the resistance of spermatozoa to the freezing and thawing process. We conclude that the individual composition of seminal plasma affects the suitability of stallions for semen cryopreservation.     

Fertility of stallion semen processed, frozen and thawed by a new procedure

The fertility of frozen-thawed and fresh semen from three stallions was compared in a trial using a randomized block design and 90 mares for 108 cycles. Semen was collected every third day, diluted to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium, and centrifuged. The cells were resuspended at 700 x 10(6) progressively motile sperm/1.0 ml of added lactose-EDTA-egg yolk extender containing 4% glycerol, packaged by placing 0.55 ml into polypropylene straws, and frozen. Semen was thawed by immersion in 75 degrees C water for 10 sec. All of the 43 ejaculates collected were frozen, but 21 were discarded because progressive sperm motility was <35% immediately after thawing or <40% after 30 min of incubation at 37 degrees C. semen from the same stallions was collected daily for inseminations with fresh semen. Semen containing 200 x 10(6) progressively motile sperm was added to 10 ml of heated skimmilk extender. Mares were inseminated daily starting on the third day of estrus or when a >/=4-cm follicle was detected, whichever came later, and continuing through the end of estrus or for nine days. Based on palpation per rectum on day 50 postovulation, the pregnancy rates from inseminations during one estrus were 50, 56 and 61% with frozen semen and 67, 67 and 61% with fresh semen (P>0.05) from the three stallions, respectively. Thus, mean pregnancy rate with frozen semen was 86% of the rate attained with fresh semen.     

Seminal traits, suitability for semen preservation and fertility in the native Portuguese horse breeds Puro Sangue Lusitano and Sorraia: Implications for stallion classification and assisted reproduction

The Puro Sangue Lusitano (PSL) is the major national breed of horse in Portugal, but no studies exist on its seminal characteristics, or on the possibility of conserving semen for future use. The aim of this study was to evaluate semen parameters, fertility and the aptness to semen preservation in Lusitano Stallions. In order to compare characteristics defined by a single or by multiple semen collections per stallion 152 ejaculates obtained from 152 Lusitano stallions presented at an annual breeding soundness examination as well as data related to 371 ejaculates obtained from 9 PSL were analyzed. These latter samples were also evaluated in terms of their possible use in assisted reproduction and were compared with 113 ejaculates obtained from 4 Sorraia horses, a rare and endangered Portuguese breed. The percentage of motile spermatozoa (PMS) was assessed after collection (AC), after semen dilution (AD) and at 24h of cool-storage. Mean values obtained for sperm motility and morphology and semen pH observed after semen collection differ significantly (P<0.05) between single collection/multiple stallions and multiple collections/limited stallions, and no age related effects were detected. Overall, Lusitano semen quality was comparable to that of related breeds, while Sorraia stallions had very poor semen quality. The response to cool-storage of diluted semen samples differed among stallions and breeds, and the best results for progressive motile sperm cells at 24h were in a range of 35-53% for PSL stallions and were lower for Sorraia stallions. Fertility rates obtained with artificial insemination (AI) averaged at 85% for PSL. With the exception of PMS AC, sperm vitality and semen pH no other seminal trait seemed to influence fertility rates in the Lusitano breed.