Sperm morphology in Estonian and Tori Breed Stallions

The standard procedure for assessing the breeding potential of a stallion includes the parameter total number of spermatozoa classified as morphologically normal. This study investigated sperm morphology of fresh semen in randomly chosen Estonian (E, n = 8) and Tori (T, n = 7) breed stallions with proven fertility. Two ejaculates were examined from each stallion. An aliquot from each ejaculate was fixed in 1 mL formol-saline immediately after collection and examined with phase-contrast microscope at a magnification 1000× for all types of morphological abnormalities. Furthermore smears were prepared and stained according to Williams (carbolfuchsin-eosin) for a more detailed examination of the sperm heads with light microscope at a magnification 1000×. Analysis of variance was applied to the data, and results are presented as LSmeans (± SE). One T stallion that had a disturbance in the spermatogenesis and one 22-year-old E stallion were not included in the analyses. The T stallions had on average 57.5 ± 4.1% and the E-stallions 74.4 ± 3.8% morphologically normal spermatozoa (p = 0.012). In 4 of 7 T stallions and 7 of 8 E stallions both ejaculates had >50% morphologically normal spermatozoa. There was a significant difference between breeds in mean percentage of proximal droplets (17.3 ± 2.7% and 2.9 ± 2.5% for T and E stallions, respectively; p = 0.003).     

Effect of semen collection method on pre- and post-thaw Guirra ram spermatozoa

In this study, we evaluated the potential effect of the method of recovery (artificial vagina or electroejaculation) on the production and quality of Guirra ram spermatozoa cryopreserved for the possible constitution of a sperm bank. In order to address this question, we evaluated the effect of semen collection method on fresh semen quality parameters, including: volume, concentration, production, microscopic analysis (abnormal sperm and intact apical ridge) and sperm motility parameters determined by CASA system. For frozen-thawed semen, we evaluated motility parameters by CASA and intact apical ridge, acrosomal status, assessed by dual staining by IP and FITC-PNA and capacitation status, assessed by M540 and Yo-pro1, using flow cytometry. The main findings from this study were: (i) that electroejaculation resulted in a lower recovery efficiency (80% of the cases), as a consequence of contamination with urine or lack of response to the electrical stimulation; (ii) the fresh seminal quality was not significantly different between recovery methods, except for the concentration of spermatozoa, but total number of spermatozoa and the consequent number of possible seminal doses for artificial insemination were similar; and, (iii) a higher number of stable and functional spermatozoa (higher number of live non-capacitated cells, higher live acrosome intact cells and live acrosome reacted cells) were found for frozen-thawed spermatozoa collected by electro ejaculation than by artificial vagina. According to our results, we are able to develop both methodologies in the creation of the Guirra sperm bank. Assuming the advantages and limitations of both methodologies, in Guirra breed, would enable the rapid constitution of a sperm bank including samples from a large number of non-trained rams in a short period of time, which will increase the genetic variability, and so guarantee the conservation of this breed.     

Zona pellucida binding and zona-induced acrosome reactions in horse spermatozoa: comparisons between fertile and subfertile stallions

Diagnostic tests that probe sperm function are needed to determine the potential etiologies of subfertility and to explore treatments of subfertility in stallions. Using epifluorescence and phase contrast microscopy, a comparison was made between ejaculates from 3 fertile and 3 subfertile stallions in which sperm-zona pellucida binding and acrosomal status were measured. Motile spermatozoa were selected by Percoll gradient centrifugation and were capacitated in vitro using TEST:TALP capacitation medium at 39 degrees C under humidified air containing 5% CO2. Concentration of motile spermatozoa was held constant during co-incubation with oocytes for fertile and subfertile ejaculates. The total number of zona pellucida-bound spermatozoa was higher for fertile stallions than for subfertile stallions (P < 0.05). Similarly, the percentage of acrosome reactions in zona pellucida-bound spermatozoa was higher for the 3 fertile stallions than for the 3 subfertile stallions (P < 0.05). These results indicate that spermatozoa from fertile stallions may interact with female gametes differently from that of subfertile stallions and suggest that sperm functions are measurable and may vary with fertility.     

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.     

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.     

Evaluation of in vitro capacitation of stallion spermatozoa

The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca(2+)-dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO(2) on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular regard to hyperactivation, was analyzed. For the study, fresh semen was washed and then incubated for 5 h in bicarbonate-containing or bicarbonate-free medium, with or without Ca(2+) ionophore to induce the AR, and at intervals during incubation aliquots were taken and analyzed for capacitation and acrosome status. The AR was assessed using both the CTC and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) staining techniques with similar results. In brief, it was found that merocyanine 540 detects capacitation-related changes much earlier than CTC does (0.5 h versus approximately 3 h), and that flow cytometry for evaluation of capacitation and AR was a quicker (10 sec per sample) and more accurate (10,000 cells counted) technique than fluorescence microscopy. Furthermore, it was observed that Ca(2+) ionophore could not induce the AR in the absence of bicarbonate, but that the ionophore synergized the bicarbonate-mediated induction of the AR as detected by CTC (although it was not significant when evaluated using FITC-PNA). The percentage of hyperactive sperm in each sample was not affected by time of incubation under the experimental conditions studied. In conclusion, merocyanine 540 staining is a better method than CTC staining for evaluating the early events of capacitation for stallion spermatozoa incubated in vitro. Furthermore, bicarbonate sperm activation clearly plays a vital role in the induction of the AR in stallion spermatozoa.     

Capacitation and acrosomal exocytosis are enhanced by incubation of stallion spermatozoa in a commercial semen extender

Preserved stallion semen often has decreased spermatozoal motility and fertility that can vary significantly between individual stallions. It is not known whether the medium used for extending equine sperm contributes to these decreases by inducing premature capacitation during storage. If spermatozoa undergo capacitation or acrosome reaction prior to insemination, this could result in a diminished capacity to penetrate the cumulus mass and fertilize the egg. We hypothesized that skim milk-based semen extenders, similar to those used in cooled storage, stabilize sperm membranes and prolong sperm motility and longevity. However, this could decrease the efficiency of sperm to undergo subsequent capacitation in vivo. This study was designed to evaluate the effects from two media on sperm function. Spermatozoal motility was analyzed, intracellular calcium was measured, and the ability of sperm to undergo acrosome reaction was compared after incubation in a skim milk extender (SME) and Tyrode's medium containing albumin, lactate, and pyruvate (TALP) at 37 degrees C. Results suggest that the SME facilitated capacitation as detected by an increase in both intracellular calcium and acrosome reactions, and a decrease in motility, as compared to TALP. Our data support a shortened functional lifespan for equine sperm in skim milk extender, which indicates that further refinements in cooled semen preservation are required to improve fertility of transported equine semen.     

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

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

Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona-free hamster eggs

The acrosome of the stallion spermatozoon was visualized by indirect immunofluorescence with monoclonal antibody (18.6) which recognized an integral acrosomal membrane component. Localization was confirmed by electron microscopy using peroxidase labelled antibody. In fresh semen samples (n = 19), 73.9 +/- 9.1% of the spermatozoa from five fertile stallions displayed a uniform bright fluorescence over their acrosome region. In two semen samples from an infertile stallion only 28% and 35% of spermatozoa showed the same pattern of fluorescence. Spermatozoa from fertile stallions incubated for up to 12 hours in TALP medium maintained motility and exhibited a significant progressive loss of acrosomes as detected by immunofluorescence. Alternatively, a similar loss of acrosomes could be induced with calcium ionophore A23187 over a 90 minute incubation. Ultrastructural observations and incubation with zona-free hamster eggs indicated that only with ionophore treatment was immunofluorescent acrosome loss correlated with a physiological acrosome reaction, while prolonged sperm incubation led to degenerative membrane changes. It was concluded that, if carefully validated, immunofluorescent localization of the acrosome of stallion sperm with monoclonal antibody could be used to monitor the acrosome reaction. Furthermore, definitive acrosome visualization would be valuable in assessing semen quality.     

The influence of age and breed on stallion semen

This study reports on the variation in semen quality and in spermatozoal and behavioral characteristics of 168 stallions representing 9 breeds and ranging in age from 2 to 26 yr. Semen samples were collected into an artificial vagina and the number of mounts and urethral pulsations per semen sample were recorded. Semen characteristics were examined for total volume, gel-free volume, gel volume, color score, mass activity, nonmotile spermatozoa, dead spermatozoa, semen density, spermatozoa concentration, total number of spermatozoa and semen pH. Morphological characteristics of the spermatozoa included abnormal heads, abnormal mid-pieces, abaxial mid-pieces, protoplasmic droplets and abnormal tails. Sources of variation were evaluated and the overall means calculated by least-squares analyses of variance for nonorthogonal data. The significance of breed effects and between stallion variability were estimated using mixed-model procedures. All semen characteristics with the exception of color and urethral pulsations had significant variation due to age. Semen quality (gel-free volume, sperm concentration, total sperm numbers and sperm abnormalities) was poorest in stallions under 3 yr of age and over 11 yr. Significant breed variation was apparent in most characteristics except for pH, semen color, abnormal midpieces and urethral pulsations. It is recommended that both the age and breed of stallion be taken into consideration when evaluating stallion semen.     

Usefulness of a triple fluorochrome combination Merocyanine 540/Yo-Pro 1/Hoechst 33342 in assessing membrane stability of viable frozen-thawed spermatozoa from Estonian Holstein AI bulls

In a situation where technology allows for the simultaneous measurement of numerous parameters of a single sperm cell, it becomes crucial to choose those parameters which may be useful in estimating in vivo fertility. Sperm membrane destabilization is believed to occur during chilling of semen, although its effect on the post-thaw (PT) fertility of the spermatozoa has not yet been fully assessed. For this reason, we tested a new combination of fluorophores, Merocyanine 540 (M540)/Yo-Pro 1/Hoechst 33342 (H33342), to detect sperm plasma membrane destabilization in bull spermatozoa conventionally processed for artificial insemination (AI). The samples were tested by flow cytometry (FC), both immediately PT and following an in vitro swimup (SU) technique, and results were thereafter compared with conventional sperm quality measurements (of concentration, motility, morphology, and membrane integrity), including in vivo fertility. Semen samples from six Estonian Holstein (EHF) AI bulls, frozen when the sires were aged 3, 5, and 7 years, allowed us to test the effect of bull age on quality of semen. Plasma membrane stability correlated to motility, normal head morphology (p<0.05), and membrane integrity (p<0.01). Following the SU selection, motility, membrane integrity (p<0.001), and membrane instability increased (p<0.01), as did stability (p<0.05). Bull age did not influence the degree of sperm membrane destabilization, except for the 3-year sample versus 7-year sample, in which the proportion of spermatozoa with destabilized plasma lemma increased PT (p<0.05) without affecting membrane integrity. Only parameters measured after SU, such as proportion of total motile and linearly motile spermatozoa, assessed with computer-assisted sperm analysis (CASA) (p<0.01), average path velocity (VAP) (p<0.001), and percentage of spermatozoa with unstable plasma lemma (p<0.05), had a significant relationship with non-return rate (NRR). The results indicate that a triple combination of the fluorophores M540/Yo-Pro 1/H33342 is suitable for monitoring the status of membrane stability in frozen-thawed (FT) bull spermatozoa. As well, a SU preselection method seems helpful in distinguishing relationships between sperm quality and fertility among bulls in a homogenous sire population.     

Fertility of frozen-thawed stallion semen extended in lactose-EDTA-egg yolk extender and packaged in 1.0-ml straws

The fertility of frozen-thawed and fresh semen from each of three stallions was compared in an experiment with a randomized block design using 128 mares. Semen was collected every third day, extended in lactose-EDTA-egg yolk extender at a concentration of 500 × 10⁶ progressively motile sperm per 1.0 ml, and frozen in individual-dose, 1.0-ml straws (1.9 mm × 267 mm). The same stallions were collected daily for inseminations with fresh semen. For each insemination dose with fresh semen, 300 × 10⁶ progressively motile sperm were added to 10 ml of heated skim milk extender. Mares were inseminated daily from the second day of estrus through the end of estrus. Of 52 ejaculates processed and frozen, 38% were discarded because < 35% of the sperm were progressively motile after thawing. Based on rectal palpations on day 50 post-ovulation, pregnancy rates for inseminations during one estrus to semen from the three stallions were 17, 33 and 35% for frozen-thawed semen and 60, 62 and 64% for fresh semen. Pregnancy rates with frozen semen from two of the three stallions were 54% of the rates attained with fresh semen.     

Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen-thawed semen

Several procedures have been proposed to assess structural and functional characteristics of cryopreserved ram semen but none so far have yielded consistent relationships with in vivo fertility. The objectives of this study were to evaluate several sperm function tests as potential markers of in vivo ram fertility (determined by pregnancy rate in ewes) using frozen-thawed semen. In experiment 1, frozen-thawed straws (n=3 per ram) of semen from three high and three low fertility rams were assessed using fluorescent microscopy for (1) progressive motility, (2) viability and, (3) acrosomal status. In experiment 2, frozen-thawed straws (n=3 per ram) of semen from 18 rams of known fertility were analysed using either computer-assisted sperm analysis (CASA) for eight motion characteristics or flow cytometric staining for: (1) viability and acrosomal status, (2) plasma membrane status and capacitation-like changes, and (3) live cells following an osmotic resistance test (ORT). In experiment 3, platelet-activating factor (PAF) was isolated from straws (n=2 per ram) of semen using high-pressure liquid chromatography (HPLC) and quantified using HPLC-tandem mass spectrometry for 18 rams. In experiment 1, no association was found between motility, viability (% live) or acrosomal status (% damaged, % intact and % reacted) and in vivo fertility. In experiment 2, no correlation was found between motility (CASA), viability (% live), acrosomal status (% live, % live intact and % reacted), capacitation status (% capacitated, % non-capacitated), plasma membrane stability (% dead) and % live cells following ORT and ram in vivo fertility. In experiment 3, there was no relationship between PAF content in spermatozoa and ram fertility. In conclusion, we were unable to relate the in vivo fertility of rams with in vitro functional tests of their frozen-thawed semen and suggest that the fertility of a given semen sample cannot easily be quantified using available in vitro tests.     

Evaluation of relative fertility of cryopreserved goat sperm

This study was designed to compare differences in the in vivo fertility of cryopreserved goat semen assessed by heterospermic insemination with differences in in vitro analyses. Five groups of does were inseminated with mixed frozen-thawed semen from different pairs of bucks. The percentage of offspring sired by each buck in the pair was compared with the relative ability of spermatozoa from that frozen-thawed ejaculate to penetrate zona-free hamster ova, relative post-thaw acrosomal integrity, ability to undergo an acrosome reaction during in vitro capacitation, and assessments of sperm motility. In 4 of the 5 different insemination pairs, the ratio of offspring born was other than 1:1. Acrosomal integrity, ability of spermatozoa to undergo an acrosome reaction, and parameters of sperm motility were not correlated with differences in relative fertility in this experiment using ejaculates from fertile bucks. The ability of spermatozoa to fuse with the oocyte plasma membrane was highly correlated with relative in vivo fertility (R(2) = 0.78, P = 0.04). This suggests that fusion with the oocyte plasma membrane is an event in the fertilization process in which significant variation exists among fertile bucks. Assessment of ability of spermatozoa to fuse with zona-free hamster ova may contribute to analysis of post-thaw fertility of frozen-thawed buck 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.     

Ubiquitination and its influence in boar sperm physiology and cryopreservation

Recent reports document the potential use of the ubiquitin protein as an indicator of mammalian sperm quality or fertility, based on poor morphology, sperm count, and other cellular qualities. However, its influence on cellular physiologic mechanisms and boar sperm cryopreservation are unknown. The objective of this research was to determine the influence of boar sperm ubiquitination (n=12 boars) on motility (using CASA), and flow cytometry and fluorescent probes (in parentheses) to evaluate mitochondrial activity (JC-1), plasma and acrosomal membrane integrity (PI and FITC-PNA), membrane fluidity (M540), and chromatin stability (TUNEL) for fresh and frozen-thawed samples. The effects of ubiquitination (determined flow cytometrically) on the ability of frozen-thawed boar sperm to capacitate (FLUO-3AM) and acrosome react (FITC-PNA) were also investigated using flow cytometry. Cryopreservation induced a decrease in the percentage of sperm that were ubiquitinated from 29 to 20% (P<0.0001), but no significant effects of ubiquitin on sperm quality (motility, membrane integrities and organization) were detected. The ability of sperm to capacitate and acrosome react was influenced by ubiquitination. Samples with more ubiquitinated boar sperm were able to maintain plasma membrane integrity (PMI) better and have fewer live acrosome-reacted cells over 120min of induced capacitation (P<0.05). In conclusion, frozen-thawed ubiquitinated boar sperm were better able to survive the physical stresses of induced capacitation, yet were still capable of capacitating and acrosome reacting, which may enable use of this assay for in the vitro evaluation of the quality of boar sperm.     

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris–egg yolk extender, equilibrated (4 °C for 4 h) and frozen at −196 °C in LN₂. The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P < 0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P < 0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P < 0.01) reduced after freezing–thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r = 0.81) and high membrane fluidity (r = 0.65), and negatively correlated with cholesterol level (r = −0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.     

Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season

The study compared quality and freezability of stallion semen during breeding and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mixed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl and skim milk). Samples of 0.5ml were cryopreserved in straws using a programmed freezer. Parameters of sperm quality were evaluated before and after freezing/thawing. These included percentages of motile spermatozoa and of morphological intact sperm. Typical injuries were demonstrated by scanning electron microscopy (S.E.M.). The acrosomal status was visualised using FITC-conjugated peanut agglutinin, and the acrosome reaction was induced by calcium ionophore A 23187. The chromatin stability was estimated by acridine orange test.In winter, the average percentages of motile and morphologically normal sperm (67 and 74.3%, respectively) were higher than during the breeding season in May (59 and 65.9%; P < 0.05). After freezing/thawing the proportions of vital and intact sperm decreased significantly. The number of motile sperm declined to 15 and 18% in May and December (range 5-40%), and of morphologically intact sperm to 51% in both seasons. Results of S.E.M. showed typical membrane ruptures in the acrosomal region and some sperm with abnormal necks. The proportion of frozen sperm with spontaneous acrosome reaction was higher during winter (86.5 versus 77.0%), suggesting a higher degree of membrane reactivity. Percentages of spermatozoa with denaturated chromatin were minimal and showed minimal differences between fresh and frozen state, stallions or seasons. An additional decondensation treatment with papain and DTE revealed a slightly enhanced number of spermatozoa with denaturable DNA after cryopreservation, especially in December (5.4 +/- 1.3%). The influence of cryoextenders was not significant for most sperm parameters, but there was a high variability between the stallions. Altogether, the influence of factors on the quality of spermatozoa has the following rank order: cryopreservation > stallion > season. Different cellular structures seem to have different susceptibilities to physicochemical stress. The cryopreservation of sperm during December results in survival rates similar to those measured during the breeding season, even more important for successful preservation is the selection of suitable semen donors.     

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.