Quality Of Frozen-thawed Semen Research Articles

Cryopreservation of ram semen in extenders containing soybean lecithin as cryoprotectant and hyaluronic acid as antioxidant.

A soybean lecithin-based extender supplemented with hyaluronic acid (HA) was assayed for effectiveness to improve the quality of frozen-thawed ram semen. HA has not been tested yet in an extender containing soybean lecithin for freezing ram semen. Thus, the aim of this study was to analyse the effects of soybean lecithin at 1% or 1.5% along with HA at 0, 0.5 and 1mgml(-1) in a Tris-based extender on the motion characteristics, membrane integrity (HOST), viability, GSH peroxidase (GSH-PX) activity, lipid peroxidation and acrosomal status after freezing-thawing. Semen was collected from four Mehraban rams during the breeding season and frozen in the six lecithin×HA extenders. The extender containing 1.5% lecithin supplemented with no HA yielded higher total motility (52.5%±1.6), viability (55.8%±1.6) and membrane integrity (44.5%±1.7), but the effects of the lecithin concentration did not reach signification. Linearity-related parameters, ALH, BCF, lipid peroxidation, GSH-PX activity, morphology and acrosomal status were not affected by the extender composition. In general, adding HA significantly decreased sperm velocity (1mgml(-1) HA), total motility (only with 1.5% lecithin), viability (1mgml(-1) HA for 1% lecithin; both concentrations for 1.5% lecithin) and membrane integrity. In conclusion, adding HA to the freezing extender supplemented with soybean lecithin failed to improve quality-related variables in ram semen. Increasing the lecithin content could have a positive effect, but further studies are needed.

The Quality of Spermatozoa of Gembrong Goats during Cryopreservation Process

Gembrong goat is an Indonesia local goat having specific characteristic that is currently categorized as a breed that is at risk of extinction. In this context, the cryopreservation of gametes is important to support a genome resource bank for storage of gametes for an indefinite period of time. Evaluation of semen and spermatozoa quality was performed to determine the survival of spermatozoa and this information will be used as a reference in the cryopreservation of semen and spermatozoa. The aim of this experiment was to study the characteristics of Gembrong goat’s semen and spermatozoa during cryopreservation process. Once a week, semen from three Gembrong goats (ages about 2-3 years old) was collected using artificial vagina and then frozen with TRIS extender. After freezing, the semen was thawed. Macro- and microscopic parameters of semen and spermatozoa were assessed in fresh and frozen-thawed semen. Results showed that in the fresh semen, the volume was 0.5 mL, sperm abnormalities was 5.74%, sperm concentration was 6731 x 106/mL, the sperm motility was 78.33%, live sperm was 83.17%, and sperm membrane integrity was 78.53%. After-thawing observation showed that sperm motility decreased to 49% (P<0.05) that was lower as compared to that in the fresh and post-equilibration semen. Similarly, the percentage of sperm viability and membrane integrity during cryopreservation showed a similar pattern with the sperm motility. In conclusion, the fresh semen of Gembrong goat had a good quality and met the requirement for further cryopreservation process. Similarly, the quality of frozen-thawed semen of Gembrong goat is eligible for artificial insemination (AI) or in vitro embryo production.

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

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

Incorporation of Ascorbic Acid, Caffeine and Chloroquine Diphosphate in Dilutor Improves Structural and Functional Status of Frozen Semen

Effect of supplementation of ascorbic acid, caffeine and chloroquine diphosphate in the dilutor on post-thaw quality of spermatozoa was studied. Thirty-two semen ejaculates (8 each from 2 Holstein Friesian and 2 crossbred; 50% Friesian x 50% Haryana) were studied. Ascorbic acid, caffeine and chloroquine diphosphate were added in Tris-egg yolk-glycerol (control) extender at the concentration of 10 mM, 0.54 mM and 7 mM, respectively. The post-thaw semen parameters studied were structural (acrosomal integrity and livability), functional [motility, per cent sperm responding to hypoosmotic swelling test, i.e. HOS positive sperm and penetrability of sperm in bovine cervical mucus, i.e. sperm penetration distance-SPD] and measurement of activities of Glutamic oxaloacetic transaminase (GOT), Glutamic pyruvic transaminase (GPT), Alkaline transaminase (AKP) and Acid transaminase (ACP). Certain enzymes in the seminal plasma. The semen from crossbred bull had significantly lower mass activity (P ≤ 0.01), initial progressive motility (P ≤ 0.01), sperm penetration distance (SPD value) (P ≤ 0.01), HOS positive sperm and seminal plasma GOT activity, but higher abnormal sperm. Incorporation of additives in the dilutor significantly improved the post-thaw semen quality, especially the post-thaw motility, post-thaw per cent live sperm, sperm penetration distance and sperm responding to hypo-osmotic solution. The leakage of all the 4 enzymes (GOT, GPT, ACP and AKP) was significantly less in the samples frozen in presence of ascorbic acid and chloroquine as compared to control. Ascorbic acid resulted in maximum improvement to the quality of frozen-thawed semen followed by chloroquine and caffeine. The study concludes that post-thaw quality of frozen semen of HF and crossbred bulls significantly improves if the dilutor is supplemented with 10 mM concentration of ascorbic acid.

The pattern of cervical penetration and the effect of topical treatment with prostaglandin and/or FSH and oxytocin on the depth of cervical penetration in the ewe during the peri-ovulatory period

Artificial insemination in sheep has two major limiting factors: the poor quality of frozen-thawed ram semen and the convoluted anatomy of the sheep cervix that does not allow transcervical passage of an inseminating catheter. It has been demonstrated that in the ewe during estrus, there is a degree of cervical relaxation mediated by ovarian and possibly gonadotrohic hormones, and we set out to investigate factors that might enhance cervical relaxation. Five experiments were conducted on ewes of different breeds to determine: 1) the pattern of cervical penetration during the periovulatory period in ewes of several breeds (Welsh Mountain, Île-de-France, Vendéenne, Romanov and Sarda); 2) the effect of the “ram effect” a socio-sexual stimulus, on cervical penetration; and 3) the effects of the intracervical administration of follicle-stimulating hormone (FSH), oxytocin and a prostaglandin E agonist (misoprostol) on the depth of cervical penetration during the periovulatory period. The results showed that during the periovulatory period in all breeds examined, there was increased penetration of the cervical canal (P < 0.05) by an inseminating catheter. Cervical penetration increased to a maximum 54 h after the removal of progestagen sponges and then gradually declined. Furthermore, the depth of cervical penetration but not its pattern, was affected (P < 0.05) by the breed of ewe. The maximum depth of cervical penetration was lower (P < 0.05) in the Vendéenne breed compared to the Île-de-France and Romanov breeds, which did not differ from one another. In the presence of rams, the depth of cervical penetration was increased at 48 and 54 h after removal of sponges (P < 0.05) and reduced at 72 h (P < 0.05). The local administration of hormones FSH, misoprostol (a PGE agonist) and oxytocin alone and in various combinations did not have any significant effect on the depth of cervical penetration during the periovulatory period. In conclusion, the natural relaxation of the cervix observed in ewes of several breeds occurs at a time during estrus, 54 h after the removal of progestagen sponges, which is the most suitable for artificial insemination. The effect was enhanced by the presence of a ram but not by the local intracervical administration of FSH, misoprostol and oxytocin even though oxytocin and PGE2 are involved in cervical function. The time of maximum cervical penetration in the preovulatory period (54 h) coincides with high LH and estradiol concentrations suggesting they might be responsible for the relaxation of the cervix probably through an oxytocin-PGE mediated pathway.

Evaluation of duck egg yolk for the cryopreservation of Nili-Ravi buffalo bull semen

This study was carried out to investigate if the substitution of chicken egg yolk (CEY) with duck egg yolk (DEY) in extenders can improve the quality of frozen-thawed semen of Nili-Ravi buffalo bulls and to study if reducing DEY level in extender affects the freezability results. Thirty semen samples collected from three buffalo bulls were diluted in extenders A, B, C, D and E containing tris, citric acid, fructose, egg yolk, glycerol and antibiotics. Extender A contained 20% CEY (control), while extenders B, C, D and E contained 5, 10, 15 and 20% DEY, respectively. After freezing and storage for 24h in liquid nitrogen, samples were evaluated for post-thaw quality.The post extension sperm motility did not differ between extenders A (control) and E (20% DEY). The same was true for post-thaw percentage of sperm with functional plasma membrane and percentage of sperm with abnormal heads or mid pieces. However, extender E showed higher (P<0.05) values for post-thaw sperm motility, livability and absolute index of livability of spermatozoa at 37°C compared to extender A. Spermatozoa with abnormal tail were lower (P<0.05) in extender E compared to extender A. Values of these parameters of post-thaw semen quality were highest for extender E containing 20% DEY and decreased significantly with decrease in the concentration of DEY, except sperm abnormalities (head, mid-piece and tail) which increased with decrease in DEY level. These results showed that replacement of 20% CEY with 20% DEY in extenders significantly improved post-thaw sperm motility, livability and absolute index of livability of spermatozoa and reduced tail abnormalities. Reduction in the level of DEY in extenders from 20% adversely affected post-thaw semen quality of Nili-Ravi buffalo bulls.

Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa

BackgroundProximal cytoplasmic droplets (PCDs), a remnant of germ cell cytoplasm, are common non-specific morphological defects in bovine semen. This study evaluated the effect of higher percentages of PCDs on the quality of frozen-thawed bovine semen, embryo production and early embryo development.MethodsThree ejaculates from each of five (group 1: PCD ≤ 1%, control) and eight adult Bos indicus bulls (group 2: PCD ≥ 24%) were analysed. Semen samples were examined for: post-thaw motility, vigour of movement, concentration, sperm morphology, slow thermoresistance test (STT), membrane integrity, acrosome status, mitochondrial function using fluorescent probes association (FITC-PSA, PI and JC-1) and sperm chromatin integrity using acridine orange assay. Two bulls from group 2, with 28.5% and 48.5% PCD, respectively, and three bulls from the control group, each with 0% PCD, were selected for IVF (in vitro fertilisation).ResultsSemen analyses revealed a significant correlation (P < 0.01) between increased rates of PCD and sperm quality traits. Nevertheless, no differences were observed in sperm motility and vigour either before or after the STT or in the percentage of intact acrosomes (analysed by differential interference contrast microscopy (DIC) after STT), but membrane integrity, acrosome status (evaluated with FITC-PSA staining method after thawing) and mitochondrial function were reduced, when compared with group 1 (P < 0.05). The higher incidence of PCD was positively correlated to chromatin damage, especially after three hours of incubation at 37°C. IVF showed similar results for bull C2 (group 1, control) and bull P2 (group 2, group with higher PCDs).ConclusionHigher PCD levels influenced spermatozoa quality traits. IVF and embryo development data showed that cleavage, blastocyst formation and blastocyst hatching may have been influenced by the interaction of morphology traits and individual bull effects.

Effect of Gamma-Oryzanol-Enriched Rice Bran Oil on Quality of Cryopreserved Boar Semen

The aim of this study was to determine the effect of gamma-oryzanol-enriched -rice bran oil on the quality of cryopreserved boar semen. Ten boars provided semen of proven motility and morphology for this study. The semen was divided into three portions in which lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with 2 types of rice bran oils, at a gamma-oryzanol concentration of 0 mg/ml of lactose egg yolk (LEY) freezing extender (group A, control), 0.1 mg/ml(0.16 mMol) of freezing extender (group B) and 0.1 mg/ml of freezing extender (group C). Semen suspensions were loaded in medium straws (0.5 ml) and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability and acrosomal integrity. There was a significantly higher percentage of progressive motility (34 versus 47.0 and 48.5, P<0.001), viability (35.5 versus 48.1 and 50.1, P<0.001) and acrosomal integrity (39.8 versus 50.8 and 54.9, P<0.001) in the gamma-oryzanol-enriched rice bran oil-supplemented groups (groups B, C) than in the control group (group C), respectively. In conclusion, addition of gamma-oryzanol-enriched rice bran oil to LEY freezing extender is appropriated for improving the quality of frozen-thawed boar semen.

The effect of antioxidants on the quality of cryopreserved semen in two salmonid fish, the brook trout (Salvelinus fontinalis) and the rainbow trout (Oncorhynchus mykiss)

Until now the supplementation of cryopreservation extenders with antioxidants has not been examined in teleost fish. Therefore, the present study investigated whether addition of antioxidants (catalase, superoxide dismutase, peroxidase, reduced glutathione, reduced methione, mixtures of reduced and oxidized glutathione or methionine) to the cryopreservation extenders could increase the quality of frozen-thawed semen of brook trout, Salvelinus fontinalis, and rainbow trout, Oncorhynchus mykiss. In brook trout and rainbow trout semen post-thaw fertility and motility were evaluated and in brook trout additionally the membrane integrity, DNA integrity, and sperm lipid peroxidation were evaluated. The tested antioxidants affected the motility parameters, DNA integrity, and fertility of cryopreserved semen, but not the membrane integrity. Most of the observed effects were negative and only minor positive effects were found. In brook trout 1.5 mmol/l reduced methionine and a mixture of 1.5 mmol/l oxidized and reduced glutathione increased the swimming velocity of frozen-thawed semen. One hundred U/l catalase, 1.5 mmol/l reduced glutathione, and 1.5 mmol/l reduced methionine slightly, but not statistically significantly increased the semen post-thaw fertility. However, these effects were not detectable in rainbow trout. Antioxidative stress or damage seems to play no role during cryopreservation, as also in the lipid peroxidation test no differences were obtained between fresh and cryopreserved semen. Therefore, for routine cryopreservation extender supplementation with antioxidants is not recommended in brook trout and rainbow trout.

Quality of frozen-thawed semen in brown bear is not affected by timing of glycerol addition

We have tested several freezing protocols for brown bear semen, modifying the time when glycerol was added (before and after cooling to 5 °C). No differences were found among protocols, indicating a good tolerance of brown bear semen to glycerol. This finding indicates that freezing protocols for brown bear semen could be modified to fit practical solutions which would facilitate preparation of the seminal samples in the field with the addition of glycerol at ambient temperature.

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.

Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of the sperm membrane and consequently a reduction in sperm motility and decreased fertility potential. The aim of this study was to determine the optimal concentration of L-cysteine needed for cryopreservation of boar semen. Twelve boars provided semen of proven motility and morphology for this study. The semen was divided into four portions in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various concentrations of L-cysteine to reach 0 mmol L(-1) (group I, control), 5 mmol L(-1) (group II), 10 mmol L(-1) (group III) and 15 mmol L(-1) (group IV). Semen suspensions were loaded in straws (0.5 mL) and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability using SYBR-14/EthD-1 staining and acrosome integrity using FITC-PNA/EthD-1 staining. There was a significantly higher (P < 0.01) percentage of progressive motility, viability and acrosomal integrity in two L-cysteine-supplemented groups (group II and group III) compared with the control. There was a biphasic effect of L-cysteine, with the highest percentage of progressive motility, viability and acrosomal integrity in group III. In conclusion, 5 or 10 mmol L(-1) was the optimum concentration of L-cysteine to be added to the LEY extender for improving the quality of frozen-thawed boar semen.

Influence of the thawing rate on the cryopreservation of semen from collared peccaries ( Tayassu tajacu) using Tris-based extenders

The objective was to evaluate the influence of the thawing rate on the quality of frozen-thawed (cryopreserved in Tris-based extenders) semen obtained from collared peccaries ( Tayassu tajacu). Semen from 13 sexually mature collared peccaries males were collected by electroejaculation, and evaluated for motility, vigor, sperm viability, membrane integrity, and sperm morphology. Semen was divided in two equal portions: the first was diluted in Tris-fructose and the other in Tris-glucose, with egg yolk (20%) and glycerol (3%) added to each portion. Extended semen was frozen in liquid nitrogen and thawed using two thawing protocols (37 °C for 1 min or 55 °C for 7 s, followed by an additional 30 s at 37 °C). There were no significant differences between the two extenders after extension, chilling, or glycerol addition. After thawing at 37 °C, there were 37.9 ± 4.2% and 28.5 ± 5.1% motile spermatozoa for samples extended in Tris-fructose and Tris-glucose, respectively, with 33.8 ± 3.7% and 28.2 ± 3.5% motile spermatozoa after thawing at 55 °C (no significant differences). Furthermore, there were no significant interactions between extenders and thawing protocols for any semen end point. In conclusion, semen from collared peccaries was successfully cryopreserved in Tris-based extenders and thawed with two protocols (37 °C for 1 min or 55 °C for 7 s).

Effect of Docosahexaenoic Acid on Quality of Cryopreserved Boar Semen in Different Breeds

During the cryopreservation process, the level of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), in the sperm plasma membrane decreases significantly because of lipid peroxidation, which may contribute to sperm loss quality (i.e. fertility) of frozen-thawed semen. The aim of this study was to investigate the effect of supplementation of DHA (fish oil) in freezing extender II on frozen-thawed semen quality. Semen from 20 boars of proven motility and morphology, were used in this study. Boar semen was split into four groups, in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various levels of fish oil to reach DHA level of 1X (group I, control, no added fish oil), 6X (group II), 12X (group III) and 18X (group IV). Semen solutions were frozen by using a controlled rate freezer. After cryopreservation, frozen semen was thawed and evaluated for progressive motility, viability by using SYBR-14/Ethidiumhomodimer-1 (EthD-1) staining and acrosome integrity by using FITC-PNA/EthD-1 staining. There was a significantly higher (p < 0.001) percentage of progressive motility, viability and acrosome integrity in DHA (fish oil) supplemented groups than control group. Generally, there seemed to be a dose-dependent effect of DHA, with the highest percentage of progressive motility, viability and acrosome integrity in group-III. In conclusion, supplementation of the LEY extender with DHA by adding fish oil was effective for freezing boar semen as it resulted in higher post-thaw plasma membrane integrity and progressive motility.

Comparison of Post-Thaw DNA Integrity of Boar Spermatozoa Assessed with the Neutral Comet Assay and Sperm-Sus Halomax Test Kit

In this study, we tested the hypothesis whether the neutral Comet assay (NCA) and the Sperm-Sus-Halomax (SSH) test kit could provide similar measurements of post-thaw DNA fragmentation of boar spermatozoa. Whole ejaculates or sperm-rich fractions of boar semen were frozen in an extender containing lactose, lipoprotein fractions isolated from ostrich egg yolk (LPFo), glycerol (lactose-LPFo-G) or in a standard boar semen extender (K3), without the addition of cryoprotective substances. In all boars, both the NCA and SSH test showed similar levels of post-thaw sperm DNA fragmentation in samples of the same ejaculates, regardless of the ejaculate collection procedure and extender. Yet, the levels of post-thaw sperm DNA damage, detected by the NCA and SSH test, were more accentuated in spermatozoa frozen in the absence of cryoprotective substances. Both the NCA and SSH detected variations among individual boars in terms of post-thaw sperm DNA fragmentation. Agreement between the measurements of the NCA and SSH was confirmed by scatter plots of differences, suggesting that the DNA integrity tests could detect the same sperm populations, which were susceptible to cryo-induced DNA damage. The findings of this study indicate that the NCA and the SSH test are effective in detecting similar levels of sperm DNA fragmentation and reinforce their importance in the assessment of frozen-thawed boar semen quality.

Effect of Different Concentrations of Ascorbic Acid on Motility, Membrane Integrity and Chromatin Status of Frozen‐Thawed Canine Spermatozoa Within Six Hours of Storage at 37°C

The aim of this study was to examine comprehensively the effect of ascorbic acid (Asc) on frozen-thawed canine semen. Pooled ejaculates (n = 10) were assessed for quality with a computer-assisted sperm analyser (CASA). After centrifugation, the semen was divided into four aliquots (A-D) of which three were diluted with Uppsala 1 extender (v/v) containing different concentrations of Asc (B: 6.8 mmol/l; C: 13 mmol/l; D: 27 mmol/l). One group without Asc served as control (A). Subsequently, dilution samples were treated and cryopreserved as described previously (Theriogenology 66, 2006, 173). After thawing, samples were stored at 37 degrees C for 1, 3 and 6 h, then examined for quality (CASA, flow cytometry, sperm chromatin structure assay; SCSA). Staining for flow cytometry was performed with FITC-PNA and propidium iodide (Reproduction 128, 2004, 829). The SCSA was performed with both Tris-NACL-EDT buffer (TNE)- and Tris-fructose-citrate buffer (TFC). In the Asc-supplemented groups, percentages of progressively motile sperm (P) were significantly lower than in the control group (A 1 h: 56.6 +/- 9.8, 6 h: 18 +/- 4.5; B 1 h: 51.2 +/- 11.8, 6 h: 13.6 +/- 5; C 1 h: 41 +/- 16.2, 6 h: 11.2 +/- 6.1; D 1 h: 37 +/- 15.2, 6 h: 8.6 +/- 5; p < 0.01), whereas, the percentages of intact cells without acrosome reactions did not differ between groups (p > 0.05). Furthermore, there were no significant differences between TNE- and TFC-samples, for alphaT, for SD of alphaT or for comp alphaT [comp alphaT: TCF-A: 2.5 +/- 1.7%, TNE-A: 3.4 +/- 3.2%; TCF-D: 2.5 +/- 2%, TNE-D: 3.3 +/- 4.3%; p > 0.05]. However, samples diluted with both extenders correlated concerning alphaT, but not comp alphaT. We therefore recommend using TNE-buffer for SCSA with cryopreserved canine semen. In addition, the average alphaT values did not differ significantly between the controls and all other groups (TNE-A: 380.2 +/- 89.1; TNE-D: 338.1 +/- 137.4; p > 0.05). It can be concluded that addition of Asc to cryoextender does not increase quality of frozen-thawed canine semen.

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing–thawing caused a significant decrease ( P < 0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower ( P < 0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics ( P < 0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.

Improvement of liquid and frozen-thawed semen quality of Nili-Ravi buffalo bulls ( Bubalus bubalis) through supplementation of fat

The aim of the study was to investigate the effects of dietary fat on quality of liquid and frozen-thawed semen of Nili-Ravi buffalo bulls. Adult bulls ( n = 21) were fed a balanced ration (Con; n = 7) or the same ration either containing sunflower oil (SF-O; n = 7) or whole sunflower seeds (SF-S; n = 7) for 63 days. Body weight and body condition score of each bull was recorded on days 0, 30 and 60 of the experiment. Semen was collected on days 39, 46, 53 and 60, frozen by a fast method and stored at −196 °C for 24 h. Sperm motility was assessed using a bright field microscope. Plasma membrane integrity of fresh and frozen-thawed spermatozoa was assessed using a hypo-osmotic swelling (HOS) assay. The concentration of spermatozoa and volume of semen was not different among groups on various days of collection. Sunflower-enriched diets did not affect the motility and number of HOS-positive spermatozoa in the fresh semen. Motility and HOS of post-thawed spermatozoa were higher ( p < 0.05) in bulls fed the sunflower-enriched diets. Similarly, diets did not affect the body condition score and body weight of bulls. In conclusion, feeding of sunflower oil or sunflower seed as fat sources can improve the quality of buffalo bull spermatozoa.

Auswirkungen der Handhabung von Pailletten während der Lagerung auf die Qualität von Stiersamen

The aim of the present study was to investigate the influence of semen handling by different inseminators on quality of frozen-thawed bovine semen. For this study a total of 10 ejaculates were collected from 7 bulls and semen stored frozen for 7 months in vessels of 10 inseminators each, in three different regions of Switzerland (A, B and C). After storage motility and acrosome integrity (live spermatozoa with intact acrosomes) were measured in frozen-thawed semen by computer-assisted sperm analysis (CASA) and flowcytometry, respectively. In addition, the 56 days non-return rate (NRR) was determined for each inseminator and the relation between NRR of single inseminators and semen quality examined. Results demonstrate that motility as well as acrosome integrity of stored frozen semen were significantly influenced by the specific delivery system used in the different regions and by the ejaculate. After storage of straws in region B semen motility was significantly (P < 0.05) lower than in region C. In region B the percentage live and acrosome intact spermatozoa as well as the NRR of the inseminators were significantly (P < 0.05) lower than in region A. Motility and acrosome integrity of semen stored by single inseminators were significantly correlated with the inseminator's NRR. We conclude that the quality of frozen semen clearly varied between inseminators of the three regions with an impact on NRR.

Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing–thawing

Whole ejaculate or sperm-rich fraction, collected from four sexually mature boars, was frozen in an extender containing lactose-hen egg yolk with glycerol (lactose-HEY-G) or extender containing lactose, lyophilized lipoprotein fractions isolated from ostrich egg yolk and glycerol (lactose-LPFo-G), and Orvus Es Paste, respectively. The sperm samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Sperm DNA integrity was assessed using a modified neutral comet assay. Sperm characteristics such as motility, plasma membrane integrity (SYBR-14/PI), mitochondrial function (rhodamine 123) and acrosome integrity were monitored. Freezing–thawing caused a significant increase ( P < 0.05) in sperm DNA fragmentation, irrespective of the procedures of ejaculate collection and extender type. Sperm DNA fragmentation was significantly lower ( P < 0.05) in the whole ejaculate compared with the sperm-rich fraction, indicating that spermatozoa maintained in the whole seminal plasma prior to its removal for freezing–thawing procedure were less vulnerable to cryo-induced DNA fragmentation. Furthermore, spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender exhibited lower ( P < 0.05) DNA fragmentation than those frozen in the absence of cryoprotective substances. The levels of sperm DNA damage, as expressed by comet tail length and tail moment values, were significantly higher ( P < 0.05) in sperm samples frozen in the absence of cryoprotective substances. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. It can be suggested that evaluation of DNA integrity, coupled with different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, may aid in determining the quality of frozen-thawed boar semen.