Use of Glutamine and Low Density Lipoproteins Isolated from Egg Yolk to Improve Buck Semen Freezing

To improve the results obtained with a reference cryopreservation extender (control extender: Triladyl® + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 m m (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 m m . In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer-assisted semen analysis. In experiment 1, glutamine at concentrations of 20 m m and 40 m m significantly improved sperm motility compared with the control extender. However, at 120 m m , a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 m m compared with the control. In experiment 3, 8% LDL and 25 m m glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (Triladyl® + 8% (v/v) LDL + 25 m m glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen.     

Application of Techniques for Sperm Selection in Fresh and Frozen-Thawed Stallion Semen

The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb^®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb^® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb^® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb^® filtration (53.6 ± 22.3%). GWS or Leucosorb^® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 10⁶ pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb^® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb^® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.     

Morphology and Chromatin Integrity of Stallion Spermatozoa Prepared by Density Gradient and Single Layer Centrifugation Through Silica Colloids

The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.     

Estimation of genetic parameters and genome scan for 15 semen characteristics traits of Holstein bulls

A QTL detection experiment was performed in French dairy cattle to search for QTL related to male fertility. Ten families, involving a total of 515 bulls, were phenotyped for ejaculated volume and sperm concentration, number of spermatozoa, motility, velocity, percentage of motile spermatozoa after thawing and abnormal spermatozoa. A set of 148 microsatellite markers were used to realize a genome scan. First, genetic parameters were estimated for all traits. Semen production traits were found to have moderate heritabilities (from 0.15 to 0.30) while some of the semen quality traits such as motility had high heritabilities (close to 0.60). Genetic correlations among traits showed negative relationships between volume and concentration and between volume and most quality traits such as motility or abnormal sperm while correlations between concentration and these traits were rather favourable. Percentages of abnormal sperm were negatively related to quality traits, especially with motility and velocity of spermatozoa. Three QTL related to abnormal sperm frequencies were significant at p < 0.01. In total, 11 QTL (p < 0.05) were detected. However, the number of QTL detected was within the range of expected false positives. Because of the lack of power to find QTL in this design further analyses are required to confirm these QTL.     

Effects of the Inclusion of Equex STM into Tris-Based Extender on the Motility of Dog Spermatozoa Incubated at 5°C

The aim of this study was to estimate the effects of Equex STM on sperm motion characteristics in chilled dog semen extended in Tris-based diluent. Thirty-two ejaculates were collected from 12 proven German shepherd stud dogs. The sperm-rich fractions were diluted in Tris-based extender with 1% (v/v) Equex STM (sample A) and in Tris-based extender with no addition of detergent (sample B). The extended semen was incubated for 240 h at 5°C and the motility parameters were evaluated by CASA system at 24-h intervals. Addition of Equex STM to Tris-based extender led to an initial activation of motion activity of spermatozoa, followed by a rapid decrease, shortening the lifespan of spermatozoa incubated at 5°C. Computer-assisted sperm analysis clearly showed that Equex STM-induced changes of sperm motion characteristics resemble the hyperactivation (HA) of spermatozoa associated with the capacitation process.     

Influence of Macrophages on the Rooster Spermatozoa Quality

The goal of this study was to evaluate the occurrence of macrophages in rooster semen and to investigate their impact on the spermatozoa quality. Ross 308 breeder males (n = 30) with no evidence of genital tract infections were used to determine the concentration of macrophages using fluorescently conjugated acetylated low‐density lipoprotein (AcLDL). Subsequently, the roosters were divided into two groups on the basis of semen macrophage concentration, and semen quality was compared in two heterospermic samples. We applied computer‐assisted semen analysis (CASA) system to determine motility parameters. Fluorescence microscopy and flow cytometry were used to evaluate occurrence of apoptotic and dead spermatozoa. Spermatozoa fertility potential was examined after intravaginal artificial insemination of hens. Eighteen roosters (control group) contained 0.2–3% of macrophages within spermatozoa population and ten roosters (macrophage group) had 10–15% of macrophages. Males from macrophage group had lower (p < 0.05) motility parameters (total and progressive movement, velocity curved line) and increased concentration of dead spermatozoa detected by flow cytometry and fluorescence microscopy (p < 0.001 and p ? 0.05, respectively). Differences (p < 0.05) between fluorescent microscopy and flow cytometry in results on spermatozoa apoptosis and viability were observed. No significant difference was found between groups in fertility of spermatozoa. In conclusion, the higher presence of macrophages in rooster semen may have a negative effect on some parameters of rooster spermatozoa evaluated in vitro. Furthermore, our study suggests that flow cytometry allows more precise examination of spermatozoa viability and apoptosis in a very short time compared with the fluorescent microscopy.     

The Relationship between Semen Quality and the Nonreturn Rate of Bulls

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The value of routine evaluation of bull semen was analysed for 117 AI bulls placed in two studs. Data from semen analysis of a total of 1635 ejaculates was compared statistically with the nonreturn rates of the bulls. The semen parameters which were significantly correlated to nonreturn rates were the motility of the freshly collected ejaculates (p = 0.0140) and post-thaw motility (p = 0.0075). The total number of motile sperm in the inseminate ranged from 10.9 to 19.3 × 10⁶ and according to previous reports the effects of low motility should be fully 'compensated' when doses above 10 × 10⁶ sperm/dose are used for insemination. In conclusion, the motility of freshly collected semen does not appear to be 'compensation' and a low percentage of motile sperm in an ejaculate may indicate other dysfunctions of the population of motile cells. Furthermore, post-thaw motility appears to correlate significantly with nonreturn rates. The largest proportion of the variation was explained by the breed of the bull and stud (42.2% of the variation), whereas the two motility parameters explained 10% of the total variation in nonreturn rates. Objective and precise evaluation of sperm motility in combination with other semen traits are needed to improve breeding efficiency. Although microscopic evaluation of sperm motility correlates with nonreturn rates of bulls, the methods are subjectively assessed and inaccurate and therefore do not allow a satisfactory prediction of fertility.     

Changes in Semen Quality in Estonian Holstein AI Bulls at 3, 5 and 7 years of Age

The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p < 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p < 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p < 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p < 0.001), total motility as measured by CASA (p < 0.01), linearly motile spermatozoa (p < 0.05) and CASA-assessed numbers of motile spermatozoa (p < 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls' life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.     

Effect of Melatonin Implantation to Sperm Donor Rams on Post-thaw Viability and Acrosomal Integrity of Sperm Cells in the Breeding and Non-breeding Season

The influence of melatonin administration to sperm donors on the freezability of ram semen and enzyme leakage through sperm cells during different steps of the cryopreservation process were evaluated in the breeding and non-breeding season. Melatonin implantation to rams in the breeding season improved post-thaw sperm viability and intact acrosome rates without influencing the motility rate (p   < 0.05). Likewise, the post-thaw alkaline phosphatase release through sperm cells was significantly lower in the melatonin-treated group in comparison with untreated controls (p   < 0.05). In the non-breeding season, melatonin administration enhanced intact acrosome rates (p   < 0.05) and reduced aspartate aminotransferase activity (p   < 0.05) post-thaw in the offseason ejaculates. Melatonin implantation twice in the breeding and non-breeding season did not produce any further improvement in the post-thaw sperm parameters in the non-breeding season ejaculates. It was concluded that melatonin administration to sperm donors improved freezability of ram semen collected from these rams and reduced enzyme leakage through sperm cells during cryopreservation.     

Combined cryopreservation of canine ejaculates collected at a one-hour interval increases semen doses for artificial insemination without negative effects on post-thaw sperm characteristics

A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1-hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane-intact spermatozoa determined by computer-assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen-thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.     

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

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

Improvement in In Vitro Fertilization Rate,Decrease in Reactive Oxygen Species and Spermatozoa Death Incidence in Rams by Dietary Fish Oil

Our aim was to evaluate the effects of fish oil feeding on sperm classical parameters, level of reactive oxygen spices (ROS), spermatozoa death incidence and in vitro fertilization (IVF) rate in rams. We randomly assigned nine rams, into two experimental groups (isoenergetic and isonitrogenous rations with constant level of vitamin E supplement): control (CTR; n = 5) and fish oil (FO; n = 4, 35 g/day/ram). Diets were fed for 70 days during the physiological breeding season. After a 21‐day dietary adaptation period, semen was collected weekly from each ram by an artificial vagina. Sperm classical parameters were determined by the computer‐assisted sperm analyzer system (CASA), and it was prepared for IVF process by swim‐up technique. These evaluations were performed during the first and last weeks of sampling. Intracellular ROS level and spermatozoa death incidence were detected by flow cytometry on a weekly basis after adaptation. Data were analysed with SPSS 15. The volume, concentration (3.6 and 2.7 × 10⁹/ml) and sperm progressive motility (60 and 48%) were significantly improved in the FO group compared with the CTR (p < 0.05). A comparison of two‐cell stage embryos following IVF in the two groups showed a significantly higher fertilization rate in the FO group (56%) compared with the CTR (49%). Superoxide anion (O₂^?) rate was significantly lower (p < 0.05) at the third week of sampling in the FO. Although the H₂O₂ rate was numerically lower in the FO group compared with the CTR, this difference was not significant. In addition, apoptosis showed a significant difference in the third week of sampling (15 and 30% for FO and CTR, respectively; p < 0.05). Overall, adding fish oil to the ram diet not only improved sperm quality and IVF results, it also could reduce oxygen‐free radicals and the incidence of spermatozoa death.     

Multivariate Cluster Analysis Regression Procedures as Tools to Identify Motile Sperm Subpopulations in Rabbit Semen and to Predict Semen Fertility and Litter Size

Computerized motility analysis (CASA) shows that four separate subpopulations of spermatozoa with different motility characteristics co-exist in rabbit ejaculates. There were significant (p < 0.01) differences in the distribution of these subpopulations among separate genetic lines, total sperm abnormalities and the percentage of altered acrosomes. Furthermore, logistic and linear multivariate regressions among several parameters of rabbit semen quality analysis were tested for use as predictive tools for the fertilizing ability of a specific artificial insemination semen sample. Logistic regression analysis rendered two mathematical, significant (p < 0.01) models: one between sperm viability and conception rate and the other between total sperm abnormalities and conception rate. Multiple linear regression analyses also yielded some significant relationships between both fertility (p < 0.001) and litter size (p < 0.05), with respect to some semen characteristics. Our results support the hypothesis that the predictive in vivo fertility use of the standard rabbit semen quality analysis coupled with a CASA determination could be reasonably achieved by applying linear and logistic regression analyses among several parameters of rabbit semen quality analysis.     

Technical note: Artificial vagina vs a vaginal collection vial for collecting semen from rams.

The time required to train rams to an artificial vagina (AV) makes collecting semen from large numbers of rams difficult. To manage this problem, we developed a glass, round-bottomed, 1.9-cm i.d. x 9.8-cm long vaginal collection vial (VCV). Three experiments were conducted to determine whether the VCV affected 1) semen volume per collection, 2) percentage of motile spermatozoa, 3) forward progressive motility score before and after extension and after freezing and thawing, and 4) our ability to collect semen from untrained rams. A soft rubber cap with a hole in the center was used to cover the VCV. A VCV was inserted into the vagina of an estrual ewe, and a monofilament line attached to the VCV was clipped to the wool near the vulva. Rams were joined with unrestrained ewes in a pen until they ejaculated into the VCV. In Exp. 1, five rams trained to an AV were used in a switchback design with four collection periods. During each period (1 d), semen was collected with an AV and a VCV. Immediately after collection, semen volume and sperm motility were quantified. Semen was extended with an aloe vera gel-based diluent at a 1:4 dilution rate, motility was quantified again, and semen was frozen. At 1 h after freezing, semen was thawed and sperm motility was quantified. Ejaculate volume (mean = 0.7 mL) and all measures of motility after collection were similar (P > 0.05) for the two collection methods. In Exp. 2, 10 rams trained to an AV were used in a switchback design with five collection periods (period = 3 d). On d 1 and 3 of each period, an AV and a VCV were used to collect semen. Collection method did not affect (P > 0.05) ejaculate volume (mean = 1.0 mL), percentage of motile cells, or forward progressive motility score. In Exp. 3, 51 untrained rams were used in a switchback design with a single collection period (2 d). Semen was collected with an AV and a VCV. Ability to collect an ejaculate and time required for collection were recorded. The likelihood of collecting semen from untrained rams was greater (P < 0.01) using a VCV (mean = 31.4%) than using an AV (mean = 9.8%). Collection method did not affect (P > 0.05) ejaculate volume (mean = 0.8 mL), percentage of motile cells, or forward progressive motility score. We concluded that a VCV could be used to collect semen from rams that are not trained for semen collection without decreasing ejaculate volume or sperm motility.     

Assessment of Fertility after Using Different Procedures to Thaw Ram Spermatozoa Frozen in Mini Straws

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In this study, fertility rates were compared after using different procedures (50°C and 70°C) to thaw ram spermatozoa frozen in mini straws. Semen from three, 1.5–2.5-year-old rams of the same breed, selected for use in an AI programme, was collected using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in 0.25-ml mini straws and frozen in a programmable freezer. Post-thaw sperm motility was assessed subjectively using a phase contrast microscope. Sperm membrane integrity was assessed with fluorescent dyes (Calcein AM/EthD-1). Statistically significant variation in the incidence of membrane integrity was found, both between rams and between freezing operations. Significant differences between the different thawing procedures used in this study were seen for membrane integrity (p < 0.01), as assessed with the fluorescent dyes (Calcein AM/EthD-1), but not for the post-thaw motility. The average fertility in this study was 39.7%, with a wide variation between freezing operations (not significant), rams (p < 0.001; 30.4, 33.3 and 64.6%) and flocks (p < 0.001, range: 14.8–61.6%). No statistically significant differences were found for the different thawing procedures, in terms of the fertility (39.0 and 40.4%, respectively) and the litter size (1.32 and 1.41, respectively). Thawing at 50°C for 9 s, instead of 70°C for 5 s, does not seem to further affect either fertility or litter size. The use of this lower temperature would facilitate the practical use of frozen–thawed ram semen under farm conditions in Sweden.     

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.     

The Effects of Centrifuged Egg Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to Freeze Miniature Caspian Horse Semen

The aim of this study was to determine the synergistic effects of centrifuged egg yolk (EY) and soybean lecithin on post-thaw Caspian horse sperm motility, morphological abnormalities, and assessment of membrane integrity. The centrifuged EY (CEY) was added at concentrations of 2% and 4% to a defined INRA plus 1.25% soybean lecithin extender used to freeze Caspian horse semen. In this experiment, ejaculates collected from each Caspian horse (n = 4) were divided into three equal aliquots and diluted in CEY 2% (INRA2), 4% (INRA4) supplemented, and without any CEY (INRA0) in INRA plus 1.25% soybean lecithin extender, respectively. Thereafter, samples were frozen and thawed following a standard protocol. Sperm cryosurvival was evaluated in vitro by microscopy assessments of post-thaw sperm motility (by means of computer-assisted semen motility analysis [CASA]), acrosomal and other abnormalities (head, mid-pieces, and tail) and plasma membrane integrity (evaluated by HOST). In Caspian stallion, semen extended with INRA2 had significantly higher CASA motility and CASA progressive motility than those extended with the rest of extenders after freezing and thawing (P < .001). There was no significant difference in path velocity (VAP), VCL, and ALH among three groups (P > .05). For straight line velocity (P < .01) and LIN (P < .001), the highest values were obtained from the INRA4 group. The highest percentages of acrosomal and other abnormalities were found in semen diluted in INRA4 (P < .001). In the group frozen INRA2, the percentage of membrane integrity was significantly higher than that of the other groups (P < .001). The use of CEY 2% in combination with soybean lecithin significantly improved Caspian horse semen freezability.     

Effect of adding different levels of caffeine in the extender on some biochemical constituents,enzymatic activities and physical characteristics of chilled and frozen ram semen

Two experiments were carried out to determine the efficiency of supplementation of ram semen extender with caffeine on chilled storage and frozen capacity of spermatozoa. In the first experiment, eighty ejaculates were collected by an artificial vagina from five adult Barki rams, aged 2–3 years and weighted 45.0 ± 2.0 kg throughout the experimental period (January to February 2017). The ejaculates were pooled and diluted (1:10) with tris‐citric egg yolk extender and were split into five groups. Group 1 served as control, whereas groups 2‐5 were supplemented with 0.1, 0.2, 0.3 and 0.4 mM caffeine. All diluted semen specimens were evaluated for physical characteristics immediately after dilution (T₀) and throughout preservation period of 48 hr at 4°C. Simultaneously, oxidative stress and indices such as total antioxidant capacity (TAC), malondialdehyde concentrations (MDA) and alkaline transaminase (AKP) concentrations and value of resazurin reduction test (RRT) were determined. In the second experiment, the raw pooled ejaculates were diluted (1:10) with glycerolated tris‐citric egg yolk extender, receiving the previously mentioned caffeine levels. The post‐thaw assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer‐assisted sperm analysis (CASA) system. The results revealed that adding caffeine to ram semen extender at low (0.1 mM) or medium (0.2 mM) levels had positive impact on both physical characteristics of ram sperm and the enzymatic activities compared to the other semen groups. Caffeine supplementation also enhanced post‐thaw sperm dynamics, which implies its potential as an exogenous antioxidant supplement.     

The benefits of cooling boar semen in long‐term extenders prior to cryopreservation on sperm quality characteristics

This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep^® Plus (AHP), Androstar^® Plus (ASP), Safecell^® Plus and TRIXcell^® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1^?/PI^?) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.     

Retracted: Reliable collection of Caspian brown trout (Salmo trutta caspius) sperm using a catheter

The traditional stripping procedure for collecting fish semen is associated with the risk of urine contamination, which may significantly affect semen quality and quantity. The use of a catheter as an alternative method for semen collection may overcome this problem. Therefore, this study compared Caspian brown trout (Salmo trutta caspius) semen parameters (i.e. sperm density, seminal plasma osmolality, motility parameters of spermatozoa analysed using computer‐assisted sperm analysis and fertility) between the traditional stripping method and the use of a catheter. All parameter values of the semen collected with a catheter were significantly higher (p < .05; density = 7.67 ± 1.02 × 10⁹ ml^?1 and osmolality = 279.28 ± 32.84 mOsm kg^?1) than those collected with stripping method (density = 4.85 ± 0.47 × 10⁹ ml^?1 and osmolality = 216.42 ± 20.75 mOsm kg^?1). Semen collected with a catheter was characterized by higher spermatozoa motility compared with sperm collected via stripping. Similarly, the fertilization ability of sperm collected with a catheter was significantly greater (p < .05) than sperm collected with the traditional stripping method. In conclusion, collection of sperm with a catheter was shown to effectively reduce urine contamination and is therefore recommended for the collection of Caspian brown trout sperm.