Effect of green buffer storage on the fertility of fresh camel semen after artificial insemination

Artificial insemination (AI) is one of the most widely used reproductive technologies, and there is considerably interest in commercializing this technology in camels. Storage of semen extender frozen (at -20 °C) is of considerable interest to scientists working with camels, as transportation of diluents at refrigeration temperature is not always possible given the hot, arid and remote conditions that dromedary camels exist in. Therefore, this study was conducted to compare the fertility of fresh camel semen, after dilution in fresh or frozen-thawed green buffer (GB), after AI into single and multiple ovulating female camels. No differences were observed in any sperm characteristics (motility, membrane integrity, acrosome integrity or morphology) when semen was diluted in fresh or frozen-thawed GB (p>0.05). Sperm motility was increased by dilution (fresh: 70.7 ± 4.9% and frozen: 68.8 ± 3.1%) compared with the motility of sperm in neat semen (35 ± 2.85%; p<0.05), and sperm motility changed from oscillatory to forward progressive after dilution. Pregnancy rates were higher (p<0.05) for single ovulating camels inseminated with semen diluted in fresh (72.7%) compared with frozen-thawed GB (27.3%), and fertilization rates were also higher (p<0.05) for multiple ovulating camels inseminated with semen diluted in fresh (83.3%) compared with frozen-thawed GB (11.1%). These results clearly demonstrate the detrimental effect of freezing and thawing semen diluent on the fertility of fresh camel semen. However, further studies are required to elucidate the mechanism responsible for this reduction in fertility. Moreover, these results demonstrate that the fertility of fresh camel semen diluted in fresh GB is high enough to be considered commercially viable.     

Liquid Storage of Goat Semen in Chemically Defined Extenders

The suitability of certain commercial and self‐made chemically defined extenders for liquid storage of goat semen was tested and the effects of storage temperatures, dilution rates and sperm washing and pH of extenders on the goat sperm during liquid storage were observed. Semen was collected from nine goat bucks of the Lubei White and Boer breeds using an artificial vagina. Each ejaculate after initial evaluation was diluted with a specific extender, cooled and stored at a desired temperature. Stored semen was evaluated for sperm motility and other parameters every 24 or 48 h of storage. The ranking order of the existing milk‐ and yolk‐free extenders in sustaining goat sperm motility was Androhep > Zorlesco > Beltsville thawing solution > the Tris–glucose medium. The new extender (mZA) which was formulated based on Zorlesco and Androhep was more suitable for goat sperm than Androhep. The mZAP extender with Bovine Serum Albumin (BSA) replaced with polyvinyl alcohol (PVA) worked as efficiently as the mZA in maintaining sperm motility, membrane integrity, acrosome intactness and capacitation status. Goat sperm motility was best maintained at 5°C during liquid preservation, but decreased significantly as the temperature increased. When semen was sixfold diluted, sperm motility was maintained longer (p < 0.05) after centrifugation, but sperm motility did not differ between the centrifuged and non‐centrifuged groups when semen was 11‐fold diluted. When the extender pH was adjusted from 6.6 to 6.04, the efficiency increased significantly in both Androhep and mZAP. A forward sperm motility of 34% was maintained for 9 days when buck semen was 11‐fold diluted and stored at 5°C in mZAP, with pH adjusted to 6.04. It is concluded that for liquid storage of buck semen, the mZA extender was more suitable than other extenders; BSA can be replaced with PVA in mZA; centrifugation to remove seminal plasma can be omitted by adequate dilution; and the storage temperature and pH of extenders affected sperm motility significantly.     

Application of computer-assisted semen analysis to explain variations in pig fertility

Sperm quality is often evaluated through computer-assisted semen analysis (CASA) and is an indicator of boar fertility. The aim of this research was to study the relationship between CASA motility parameters and fertility results in pigs. Insemination records and semen parameters from a total of 45,532 ejaculates collected over a 3-yr period were used. The statistical model for analysis of fertility data from these inseminations included factors related to sow productivity. The boar- and semen-related variance (direct boar effect) were corrected for the effects of individual boar, genetic line of the boar, age of the boar, days between ejaculations, number of sperm cells in an ejaculate, number of sperm cells in an insemination dose, and AI station. The remaining variance was analyzed if semen motility parameters had a significant effect. This analysis revealed significant (P < 0.05) effects of progressive motility, velocity curvilinear, and beat cross frequency on farrowing rate (FR). Total motility, velocity average path, velocity straight line, and amplitude of lateral head displacement affected (P < 0.05) total number of piglets born (TNB). Boar- and semen-related parameters explained 5.3% of the variation in FR and 5.9% of the variation in TNB. Motility parameters, measured by CASA, explained 9% of the boar- and semen-related variation in FR and 10% of the boar- and semen-related variation in TNB. Individual boar and genetic line of the boar affected (P < 0.0001) the variation in FR and TNB. No differences (P > 0.05) were observed between effects of AI stations on fertility outcome, underscoring the objectivity of the CASA system used. Motility parameters can be measured with CASA to assess sperm motility in an objective manner. On the basis of the motility pattern, CASA enables one to discriminate between the fertilizing capacity of ejaculates, although this depends on the genetic line of the boar used in AI stations.     

Comparative quality assessment of buffalo (Bubalus bubalis) semen chilled (5°C) in egg yolk- and soya milk-based extenders

Egg yolk-Tris is most commonly used semen extender; however, its use involves hygienic risk, interference with fertility and poor microscopic examination. Therefore, replacement of egg yolk with a plant-based component with protective effects on spermatozoa would be advantageous. In present study, we observed effect of soya milk-based extenders on dilution and liquid preservation of Murrah buffalo bull semen at 5°C up to 72 h in comparison with conventional egg yolk-Tris extender (Ext.1). In experiment one, a total of 32 buffalo semen ejaculates from four animals were extended and preserved at 5°C for 72 h in soya milk-based extender (Ext.2) with different percentages (10%, 15%, 20%, 25% and 30%) of soya milk for optimization of soya milk concentration. Semen quality was assessed for individual motility, viability, membrane integrity and acrosome integrity at 0, 24, 48 and 72 h of liquid preservation. The results of experiment one indicated that 25% soya milk is an optimum concentration for buffalo bull semen extender preparation. A modified method was used to prepare another soya milk-based extender (Ext.3). In the second experiment, two soya extenders (Ext.2 and 3) with optimized concentration (25%) of soya milk were comparatively assessed with egg yolk-Tris extender (Ext.1) for semen quality parameters at 0, 24, 48 and 72 h of liquid preservation. The individual sperm motility at 0 and 24 h following dilution were found non-significant among extenders. However, after 48 h of dilution, individual motility in Ext.3 was observed significantly (p < 0.05) higher than Ext.1. After 24, 48 and 72 h of dilution sperm membrane integrity in Ext.3 was found significantly (p < 0.05) higher than Ext.1. Overall, comparative evaluation of sperm parameters obtained revealed that Ext.3 containing 25% soya milk can be used as a substitute of egg yolk-based extender for buffalo semen liquid preservation.     

Effect of chilling temperature on the long-term survival of rabbit spermatozoa held either in a tris-based or a jellified extender

As the preservation of the fertilizing capacity of rabbit spermatozoa for several days after semen collection remains a major target for the artificial insemination programs of rabbit breeding, a study was conducted to compare the efficacy of 5 or 15°C as holding temperature in lengthening the preservability of rabbit semen quality during 192 h of storage both in a solid (Cunigel) and a liquid (Tris-Citric acid-Glucose; TCG) extender. Six pooled semen samples (two ejaculates/male; two-three males/pool) were taken and made four aliquots: two aliquots were tenfold diluted with the TCG extender, whereas the other two were tenfold diluted with the Cunigel extender. One aliquot per diluent was stored at 5°C and the second one at 15°C. Sperm motility (light microscope), viability (SyBr-PI staining), plasma membrane functional integrity (Hypo-osmotic swelling test) and acrosome integrity (PSA-FITC staining) were recorded at 0, 48, 120 and 192 h of storage. In liquid-stored spermatozoa, mass motility and viability were significantly higher (p ≤ 0.05) in samples stored at 5°C than at 15°C at all the storage times; at 5°C resulted also higher (p ≤ 0.05) the percentages of both forward motility at 48 h and sperm functional integrity at 120 and 192 h of storage, whereas chilling temperature did not affect acrosome integrity. With the Cunigel extender, all the semen qualitative parameters were significantly higher in sample stored at 5 than 15°C over storage time (p ≤ 0.05); only acrosome integrity at 192 h was not different according to the chilling temperatures. In conclusion, 5°C were better than 15°C for the long-term storage of rabbit semen both in the TCG and Cunigel extender.     

Cryopreservation of Dog Semen in a Tris Extender with 1% or 2% Soya Bean Lecithin as a Replacement of Egg Yolk

Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal‐protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris‐based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5‐ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC‐PNA/PI) and sperm membrane integrity (C‐FDA) were evaluated 5 min post‐thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin‐based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin‐based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin.     

Post-thaw viability of european bison (Bison bonasus) semen frozen with extenders containing egg yolk or lipids of plant origin and examined with a heterologous in vitro fertilization assay.

Basic characteristics of European bison (Bison bonasus) semen were described and the efficacies of two extenders-Triladyl, containing egg yolk, and a synthetic extender, containing soybean lipids-were tested for semen cryopreservation. Seven ejaculates were collected by electroejaculation from a 10-yr-old, European bison bull. Each ejaculate was diluted at 37 degrees C to a final concentration of 200 x 10(6) sperm/ml with Triladyl or the synthetic extender. Extended semen samples were frozen according to a standard bull semen freezing protocol. After 2 wk of storage, one straw from each extender and ejaculate was thawed, and postthaw quality was evaluated by individual sperm motility and movement rate, numbers of sperm morphologic abnormalities and intact acrosomes, functional integrity of the sperm membranes determined by hypoosmotic swelling test (HOST), viability (live-dead, eosin-nigrosin stain), and a heterologous in vitro sperm penetration assay (SPA). A total of 600 in vitro-matured bovine oocytes were inseminated with 1 X 10(6) spermatozoa of Holstein semen frozen-thawed in Triladyl (control) or of European bison semen frozen in Triladyl or the synthetic extender. Nuclear status of the oocytes was determined after 18 h of sperm-oocyte coincubation. Extender had no effect on any evaluated parameters of semen after dilution and cooling (4 hr at 5 degrees C) or in postthaw individual motility, quality of movement, and sperm morphology. However, significantly (P < 0.05) higher numbers of spermatozoa with intact acrosomes, intact membranes (HOST), and viable sperm (P < 0.01) were in semen frozen in Triladyl than in the synthetic extender. Mean values for heterologous SPA for bull (control) and for bison semen frozen in the synthetic extender were very much alike-63.3+/-10.6% and 63.1 +/- 15.9%, respectively; bison semen frozen in Triladyl was lower, 43.0+/-24.2% but not significantly different. Cumulative results from a variety of viability assays of diluted/cooled and frozen-thawed semen, including the heterologous SPA, suggest that European bison semen can be successfully frozen in both extenders tested in this study.     

Effects of Seminal Fluid Fractions on Plasma and Acrosome Membrane Integrity and Mitochondrial Membrane Potential Determined by Flow Cytometry in Chilled Canine Spermatozoa

Depending on the mammal species, the use of seminal plasma during semen processing for cryopreservation has been found to have both beneficial and detrimental effects. This study was designed to determine the effects of the second (SF) and third [prostatic fluid, (PF)] ejaculate fractions on plasma membrane and acrosome integrity, mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) translocation and sperm motility in chilled canine spermatozoa by flow cytometry. After pooling the second sperm‐rich fraction of ejaculates from six dogs, samples for each assay were preserved at 5°C for 72 h in egg yolk‐TRIS extender (EYT) alone (control) or supplemented with seminal fluid from the second (EYT‐SF) or third (EYT‐PF) ejaculated fractions. After cold storage, groups EYT‐SF and EYT‐PF showed significantly higher percentages of sperm cells with an intact acrosome [68.8 ± 1.4%, 69.6 ± 2.6% (p < 0.01)] and intact plasma membrane [48.1 ± 2.8%, 50.4 ± 8.2% (p < 0.001)] than that observed in EYT [51.7 ± 3.2% and 33.3 ± 4.1% respectively]. Only in EYT‐SF was PS translocation significantly reduced compared to EYT‐PF and EYT [3.9 ± 0.4%, 10.2 ± 2.2% and 9.0 ± 1.5%, respectively (p < 0.001)]. However, significantly diminished sperm motility was observed in EYT‐SF and EYT‐PF compared to EYT [36.8 ± 2.1%, 35.5 ± 2.3% and 78.4 ± 4.7% (p < 0.001)]. No significant differences were detected in ΔΨm (p > 0.05). In conclusion, supplementing semen extenders with seminal fluid from the second or third fractions of the ejaculate supplementation helps to preserve the integrity of the plasma and acrosome membranes along with the mitochondrial membrane potential but seems to compromise the motility of canine spermatozoa chilled for 72 h.     

Comparative Study on Five Different Commercial Extenders for Boar Semen

Increasing interest in a longer preservation of diluted boar sperm raises questions in the field concerning the choice of the extender. The aim of this study was to evaluate the longevity of boar sperm extended in currently used commercial semen extenders. Three long-term extenders and two short-term extenders were compared for different semen quality parameters that can be assessed under routine laboratory conditions. Sperm morphology, motility, pH and bacteriological contamination were investigated during a 7-day period. The number of dead spermatozoa did not differ significantly among the extenders (p > 0.05). Sperm motility was not only related with storage period but most of all with pH, especially in long-term extenders. Differences between the different extenders were prominent (p < 0.05); the sperm preserved in only one long-term extender showed good motility during the whole test period. In all cases, the pH of the extended semen increased by 0.3-0.5 in the first days of storage and was significantly correlated with a decrease in motility. Bacteriological quality had no significant influence on motility or pH of the semen. In conclusion, we can state that in both short-term extenders and in only one long-term extender, sperm longevity, as evaluated by the parameters used in this study, was sufficient during the preservation period. To preserve the quality of diluted boar semen during long-term storage, the choice of the long-term extender is important. In addition, the monitoring of the pH of extended boar semen in our study emphasizes the importance of the buffering capacity of semen extenders.     

Factors influencing boar sperm cryosurvival

Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that 70% of total variance observed in postthaw sperm quality variables among ejaculates was explained by boar. This indicates that boar is the most important (P < 0.001) factor explaining the variability among ejaculates in sperm cryosurvival, with most (14 of the 15 boars in Exp. 3) showing consistent (P > 0.05) sperm cryosurvival over time.     

Effects of Thawing Temperature and Post‐thaw Dilution on the Quality of Cat Spermatozoa

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70°C) and to investigate the effects of post‐thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25‐ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37°C for 15 s, (ii) at 37°C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70°C for 6 s, (iv) at 70°C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR‐14/EthD‐1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 ± 10.7 (mean ± SD), a score of progressive motility of 4.0 ± 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 ± 12.1 and intact acrosome of 44.8 ± 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70°C and post‐thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post‐thaw incubation.     

Breed of Boar Influences the Optimal Concentration of Gamma‐oryzanol Needed for Semen Cryopreservation

The aim of this study was to investigate the influence of boar breed on the optimal concentration of gamma‐oryzanol on the qualities of cryopreserved boar semen. Semen was collected from 20 boars (10 Duroc, 5 Large white and 5 Landrace boars). The semen sample was divided into five groups (A–E) according to the concentration of gamma‐oryzanol in extender II, that is 0, 0.08, 0.16, 0.24 and 0.32 mm , respectively. The semen was cryopreserved by nitrogen vapour and storage in nitrogen tank (?196°C). After storage for a week, samples were thawed at 50°C for 12 s and evaluated for progressive motility, sperm viability and acrosome integrity. The results demonstrated that gamma‐oryzanol significantly improved progressive motility, viability and acrosome integrity of frozen–thawed boar semen. Considering the influence of breeds on the optimal concentration of gamma‐oryzanol, for Duroc boar, gamma‐oryzanol at 0.16 mm (group C) yielded the highest percentage of progressive motility, sperm viability and acrosome integrity. For Large white and Landrace boars, gamma‐oryzanol at 0.24 mm (group D) showed a significantly higher percentage of progressive motility, viability (not significant in Landrace) and acrosome integrity than other concentrations. In conclusion, the optimal concentration of gamma‐oryzanol needed for boar semen cryopreservation in lactose–egg yolk (LEY) freezing extender is not only depended on individual boar but also breed of boar, that is 0.16 mm for Duroc and 0.24 mm  for Large white and Landrace.     

Rotation of Boar Semen Doses During Storage Affects Sperm Quality

It is common practice to rotate boar semen doses during storage for prevention of sperm sedimentation. In this study, the effect of rotation of boar semen doses during storage on sperm quality was investigated. Manual turning twice daily and automatic rotation five times per hour resulted in the following effects: alkalinization of the BTS‐extender, loss of membrane integrity at day 3, and loss of motility and changes in sperm kinematics during a thermoresistance test at day 5. Using a pH‐stabilized variant of BTS extender, sperm motility and velocity decreased in continuously rotated samples, whereas membrane integrity and mitochondrial activity remain unaffected. It is concluded that rotation of semen samples adversely affects sperm quality and, therefore, should no longer be recommended for AI practice.     

Optimization of glycerol concentration and freezing rate in the cryopreservation of ejaculate from brown bear (Ursus arctos)

In order to establish a semen bank for the endangered Cantabrian brown bear, we tested five glycerol concentrations and three freezing rates for electroejaculated semen. Electroejaculation was performed on nine males. Semen was diluted in TES-Tris-Fructose (20% egg yolk, 2% EDTA, 1% Equex) with 2%, 4%, 6%, 8% or 10% glycerol and frozen at -10, -20 or -40°C/min. Before and after cryopreservation, samples were analysed for motility (CASA), viability and acrosomal status (flow cytometry). Pre-freezing results showed that glycerol concentration had no significant effect on total motility or progressive motility, but it significantly decreased VCL, ALH, viability and acrosomal status (p < 0.05). After thawing, sperm motility was higher at extender with 4%, 6% and 8% glycerol, but only at 4% and 6% glycerol for viability and acrosomal status. For 4% and 6% glycerol, freezing rates did not have significant effects. The curve fitting gave an estimate of the optimal glycerol concentration, with all the optimal values for every parameter between 6% and 7% glycerol falling. We propose using 6% glycerol and a freezing velocity of -20°C/min for freezing brown bear ejaculated spermatozoa.     

Efficiency of three boar sperm enrichment techniques

The objective of the study was to investigate the efficiency of three enrichment methods to separate boar spermatozoa. Twenty-four ejaculates from 12 boars (2 ejaculates/boar) were extended (30 × 10⁶ spermatozoa/mL) in commercial Beltsville Thawing Solution. Each semen sample was processed with glass wool column (GW) and glass beads (GB) filtration and with the single-layer centrifugation (SLC) technique. Semen samples before (control; C) and after treatment were evaluated for sperm CASA motility/kinetics and concentration, viability, morphology and chromatin integrity. Data were analysed with mixed models. The concentration of total and motile spermatozoa was significantly decreased after treatment in groups GW and SLC, but not in group GB. Group GW showed increased values of WOB compared with both groups C and GB. Group GB showed greater values of rapid movement spermatozoa and lower values of slow movement spermatozoa compared with group C. In group SLC, higher values of VSL, LIN and STR were observed compared with group C. In conclusion, all techniques under examination enhanced various CASA variables. Based on our results, the GB method is a promising alternative separation technique for boar sperm and deserves further research regarding swine in vitro fertilization.     

Preservation of boar semen: An update

In the pork industry, artificial insemination and the storage of boar semen in liquid at 17°C are routinely applied to optimize the ejaculate and bring about rapid genetic changes that are reflected in the animal protein. Although the results are satisfactory, they are below what occurs with natural mating. It is currently possible to preserve boar semen with storage at 17°C and slow freezing, since to date there is only one study on vitrification, with negative results applicable only in the case of implementing an intracytoplasmic sperm injection. In both methods and due to the sensitivity of boar sperm to osmotic and temperature changes, there is a loss in the quality of the initial sample; however, slow freezing in boar semen has greater deleterious effects on the sample that are reflected in the pregnancy rates and number of live births. Therefore, only 1% of all inseminations are done with frozen semen. The aim of this review is to provide advances and results of studies conducted on the preservation of boar semen, delving more deeply into the critical points that each of the preservation techniques presents, including bacterial contamination, extender components, temperature, ice nucleation, use of additives in extenders and the main deleterious effects on sperm quality.     

Effects of Egg Yolk and Cooling Rate on the Survival of Refrigerated Red Deer (Cervus elaphus hispanicus) Epididymal Spermatozoa

Egg yolk is a common component to sperm refrigeration for most of the deer species, the role of which is to protect sperm membranes against cold shock. In addition, there have been many studies of conservation of ejaculated semen from stags, but few have been reported for epididymal spermatozoa. This work was designed to investigate the combined effects of cooling rates (slow: 0.23 degrees C/min vs rapid: 4.2 degrees C/min) from room temperature to 5 degrees C, and egg-yolk concentration (0, 5 or 20%) in the extender on the survival of Iberian red deer epididymal spermatozoa refrigerated at 5 degrees C. Heterospermic sperm samples were diluted to a final sperm concentration approximately 400x10(6) sperm/ml with a Tris-citrate-fructose (TCF)-egg-yolk diluent. Sperm quality was in vitro judged by microscopic assessments of individual sperm motility [sperm motility index (SMI)], and of plasma membrane (hypo-osmotic swelling test) and acrosome (NAR) integrities. Our results first showed that the presence of egg yolk in the extender significantly improves (p=0.01) the viability and sperm motility after sperm dilution. In addition, acrosome and plasma membrane integrities post-refrigeration did not differ significantly between cooling procedures; however, the SMI differed significantly between cooling procedures (slow: 46.6% vs rapid: 50.0%; p=0.01). Our results also showed that sperm quality was significantly (p<0.01) affected by the combined effects of egg-yolk concentration and cooling procedure, being rapid cooling with 20% of egg yolk the most suitable combination for epididymal sperm refrigeration. In conclusion, egg-yolk improved red deer epididymal spermatozoa characteristics after dilution. Rapid cooling protocol using TCF with 20% egg-yolk significantly improved sperm motility of red deer epididymal spermatozoa after cooling.     

Comparison of two different centrifugation extenders for preservation of frozen equine semen

This study compares a commercial semen extender (control group) to ultra high temperature (UHT) skimmed milk (treatment group) used during centrifugation for subsequent cryopreservation of equine semen. Following post‐thawing of semen samples parameters measured included motility, sperm motion kinetics (using computerised assisted semen analysis) as well as acrosome and plasmatic membrane integrity (using fluorescent dyes). After collection and analysis, the sperm‐rich fraction was divided and diluted with either: control (1:1 dilution in a skimmed milk‐glucose extender) or treatment (1:1 dilution in UHT skimmed milk). The milk used in this experiment was of the same source, commercial brand, of only one lot. After dilution, samples were subjected to centrifugation at 600 g for 10 min and sperm pellets were resuspended in a freezing extender to a concentration of 200 × 10⁶ cells/ml. Aliquots were packed into 0.5 ml straws placed in a stainless steel support and kept inside the refrigerator (5°C) for 20 min. Subsequently, these straws were placed at a height of 6 cm over liquid nitrogen for 20 min in an isotherm box. No significant differences were observed in total sperm motility (42.71 vs. 38.29%), progressive sperm motility (12.29 vs. 7.86%), plasma membrane integrity (53.43 vs. 60.14%) or acrosomal membrane integrity (93.29 vs. 93.71%) with a P>0.05 calculated between the control and the treatment groups, respectively. Considering that UHT skimmed milk has a lower cost than the commercial semen extender, this could be an option used during the centrifugation protocol to decrease the expense of the equine semen cryopreservation process and increase shelf life.     

Refrigerated Storage of Red Deer Epididymal Spermatozoa in the Epididymis, Diluted and with Vitamin C Supplementation

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 × 10⁶ sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 m m vitamin C. The remaining epididymides and the diluted samples were stored at 5°C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.     

Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws

Experiments were conducted to study the effect of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa after cryopreservation in straws. Semen (split ejaculate) in maxi-straws (6 mm o.d.) was frozen using a programmable freezing chamber. Three methods for in vitro sperm evaluation were used: motility (MOT), acrosome integrity (NAR) and flow cytometric analysis of sperm treated with carboxyfluorescein diacetate and propidium iodide to assess sperm plasma membrane integrity (PMI). No interactions were found among the three variables evaluated. Length of prefreeze exposure to glycerol, ranging from .5 min to 75 min, had no effect on post-thaw sperm viability. Exposure of sperm to a glycerol-containing extender medium at 5 degrees C gave improved post-thaw viability over that exposed at 0 degree C (P less than .05). Glycerol at a concentration of 3 or 4% resulted in maximum post-thaw MOT. Acrosome integrity values were greatest for 2 and 3% glycerol, whereas PMI was greatest when glycerol concentration was 4 to 6%. The primary cryoprotective effect of glycerol on boar semen may be extracellular. It is concluded that 3 or 4% glycerol gives maximum viability of frozen-thawed spermatozoa when the present methods are employed.