Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender

The aim of this study was to evaluate the effects of ergothioneine and cysteamine as antioxidant supplements in a soybean lecithin extender for freezing ram semen. Twenty-four ejaculates were collected from four rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and cysteamine or ergothioneine (2, 4, 6 or 8 mM). Motility by CASA, viability, plasma membrane functionality (HOS test), total abnormality, lipid peroxidation, glutathione peroxidase (GPx) activity and capacitation status (CTC staining) were assessed after thawing. Using 6 mM of either antioxidant improved total motility. Cysteamine at 6 mM and ergothioneine at 4 and 6 mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). Both antioxidants improved membrane functionality significantly, except at 8 mM. Progressive motility, kinematic parameters, GPx activity, capacitation status and sperm abnormalities were not influenced by the antioxidant supplements. In conclusion, cysteamine at 6 mM and ergothioneine at 4 or 6 mM seem to improve the post-thawing quality of ram semen cryopreserved in a soybean lecithin extender.     

Incorporation of taurine and hypotaurine did not improve the efficiency of the Uppsala Equex II extender for dog semen freezing

The working hypothesis of the present study was that supplementation of the Uppsala Equex II (UE) extender with the amino acid (AA), taurine (T) and hypotaurine (H) would improve dog sperm post-thaw quality, as previously seen for ram and bull semen, respectively. Five pools from 15 ejaculates of 15 dogs were used. Each AA was added to the UE extender at a concentration of 25, 50 and 7 5mM. Amino acid-free extender was used as a control. The following post-thaw parameters were evaluated: sperm motility by light microscopy and by CASA evaluation, longevity, viability (eosin-nigrosin staining), and flow cytometry (FC) was used to assess acrosome integrity and mitochondrial activity after PI/Fitc-PSA and PI-Rhodamine staining, respectively. Post-thaw sperm motility and velocity did not differ among extenders. Amplitude of lateral head displacement was lower for sperm frozen in the 25 mM H-supplemented extender. Semen frozen in the extender with 50 mM of T resulted in higher number of live sperm with damaged acrosomes after thawing. Higher numbers of live sperm with minimal mitochondrial activity were obtained for samples frozen with 25 and 50 mM T-supplemented extenders. Semen frozen in the control and 50 mM T-supplemented extenders had the highest number of live (eosin-nigrosin stain negative) sperm immediately post-thawing. We concluded that supplementation of the Uppsala extender with T or H did not improve sperm post-thaw mitochondrial activity or semen motility and viability.     

A dextran swim-up procedure for separation of highly motile and viable ram spermatozoa from seminal plasma

Although washing of sperm cells by centrifugation is a procedure in widespread use, there have been indications that centrifugation may be harmful to the cells. The objective of this study was to develop a modified swim-up technique, without centrifugation, to get a selection of highly motile and viable ram spermatozoa free of semen plasma.Semen collected from 3 rams over a period of a year was pooled into a low, medium and high motility group, and aliquots from each pool were placed beneath a dextran solution and overlaid with medium. The top layer of the medium was collected (and replenished) 4 times at 15-min intervals. Evaluated were the pre- and post-swim-up progressive individual motility, membrane integrity and resistance to a hypoosmotic swelling test (HOS). Semen samples with initial motility < 60% showed the highest relative improvement in all 3 parameters; samples with 65 to 70 and > 70% initial motility improved less but showed final absolute values similar to those in the low motility group and to each other. The first swim-up layer had the highest contamination with semen plasma (17% beta-N-acetylglucosaminidase (NAG), 13% citric acid content) and the lowest motility score. The second, third and fourth fractions were pooled and showed low plasma contamination (2% NAG and 5% citrate), 80% motility, 70% HOS, and 72% viability, up from the pre-swim-up values of 68, 66 and 59%, respectively. Our data suggest that the dextran swim-up procedure is suitable for evaluating ram spermatozoa for in vitro and in vivo procedures in assisted reproduction.     

Extending the interval between second vaccination and slaughter: II. Changes in the reproductive capacity of immunocastrated ram lambs

Immunocastration improves the welfare of castrated commercial slaughter lambs; however, the time-point at which this technique influences semen quality and sperm production has not yet been established for various vaccination schedules. Furthermore, the effect of extended intervals between second vaccination and slaughter needs to be investigated regarding continued testosterone suppression in immunocastrated lambs. The effect of extending the interval between second immunocastration vaccination and slaughter from four to six weeks on the reproductive capacity of Dohne Merino lambs was examined. A total of 40 Dohne Merino lambs were stratified according to initial weight (45.4±3.68 kg) and randomly assigned to four treatments that included intact control rams (R), Burdizzo-castrated lambs (B) and lambs immunocastrated with either four (ICS4) or six (ICS6) weeks between second vaccination and slaughter. Blood and semen samples were collected throughout the study period to determine serum testosterone concentrations, evaluate semen quality and assess sperm viability. Semen samples from R showed improvement over the trial. Throughout the collection period, B lambs had low serum testosterone concentrations, poor sperm motility and sperm viability, as expected. However, a slight increase in the percentage of live sperm in semen samples from B lambs towards the end of the collection period indicated poor success rates of the technique in some lambs. Burdizzo-castration also caused testes tissue necrosis and abscessing, indicating physiological stress. Semen appearance scores varied for both immunocastrated treatments, but the mass motility scores decreased over time. The ICS6 lambs showed a consistent and continuous decline in serum testosterone concentrations and sperm viability, with an increased percentage of dead abnormal sperm in the semen samples at the end of the study. The ICS4 treatment was successful in interrupting serum testosterone production and reducing semen quality; however, not as consistently as the ICS6 treatment. Primary immunocastration vaccination influenced serum testosterone concentrations but consistently low levels were only realised for both treatments after secondary vaccination. Although all castration treatments influenced testes size and colour, the six-week vaccination-to-slaughter interval caused a greater decrease in testes cut surface L* (lightness) colour values and in seminiferous tubule circumference. Extending the interval between second immunocastration vaccination and slaughter resulted in a more consistent and reliable influence on reproductive capacity of immunocastrated lambs. Thus, immunocastration is a suitable alternative to Burdizzo-castration regarding the interruption of testosterone production and testis functioning.     

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

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

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 degrees C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 degrees C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.     

Assessment of ram sperm membrane integrity following different thawing procedures

Semen from five 2.5-yr-old rams selected for use in an AI program was collected over 3 consecutive days using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in French mini-straws (approx. 100 mill/straw), and frozen in a programmable freezer. Three freezing operations were carried out per ram. Three straws per freezing operation were subjected to the following thawing procedures: 1) 70 degrees C, 5 sec; 2) 50 degrees C, 9 sec and 3) 35 degrees C, 12 sec. Post-thaw sperm motility was subjectively assessed using a phase contrast microscope; while the combined fluorochromes carboxyfluorescein diacetate and propidium iodide (CFDA/PI), the hypo-osmotic swelling test (HOS) and the presence of normal apical ridges (NAR's) were used to determine the degree of sperm membrane integrity. Significant differences between thawing treatments were found for post-thaw motility (P < .05) and membrane integrity (P < 0.01), and variation among rams was statistically significant. Post-thaw sperm motility as well as the percentage of spermatozoa showing intact membranes were significantly higher (P < 0.01) for straws thawed at 70 degrees C than for those thawed at 35 degrees C (67.0 +/- 1.1 and 63.0 +/- 1.1%, and 50.5 +/- 1.5 and 41.7 +/- 1.5%, respectively). However, no corresponding statistically significant difference could be found for these parameters when 70 degrees C and 50 degrees C thawing were compared. It was concluded that sperm can be thawed at 50 degrees C for 9 sec instead of 70 degrees C for 5 sec without further reducing sperm motility or membrane integrity. This lower thawing temperature would facilitate the widespread use of frozen/thawed ram semen under farm conditions in Sweden.     

Use of chromatin stability assay, mitochondrial stain JC-1, and fluorometric assessment of plasma membrane to evaluate frozen-thawed ram semen

Cryopreservation of semen imposes deleterious effects on spermatozoa, either killing a certain proportion of cells or causing subtle damages on sperm function in the surviving population, changes not easily revealed by conventional assays. We have tested three functional assessment techniques in frozen-thawed ram semen from six adult rams, cryopreserved following eight different protocols (four extenders, and glycerol being added at two temperatures). Semen samples were thawed and the following analyses were carried out: motility (CASA), membrane integrity (Hoescht 33258 and fluorometry), chromatin status (chromatin stability test and fluorescence-assisted cell sorting, FACS) and mitochondrial activity (JC-1 and FACS). Fluorometry outcome did not correlate with the other parameters and showed large variation, albeit discriminating among cryopreservation techniques (P < 0.01). Mitochondrial activity correlated, but with low values, with total and progressive motility. However, good sperm motility and high velocity values were associated to high mitochondrial membrane potential. The chromatin stability assay was also successfully carried out, and had a good relationship with male factor (%COMP alpha(t) and SD alpha(t) parameters). In conclusion, fluorometric assessment of membrane integrity albeit rendering poor results, merits improvement, being a low-cost and handy technique, especially for work in the field. On the other hand, both assessments of chromatin stability and mitochondrial status (JC-1 staining), combined with FACS, are reliable techniques that can be used for the functional assessment of frozen-thawed ram semen.     

Use of antioxidants reduce lipid peroxidation and improve quality of crossbred ram sperm during its cryopreservation

Ram sperm are subjected to extreme oxidative stress during their preservation at −196 °C resulting in reduced quality at post thaw. Therefore, the main objective of this study was to evaluate the effect of antioxidants taurine, quercetin and reduced glutathione on the post thaw quality of crossbred ram sperm. A total of twenty four ejaculates from six crossbred rams were collected and extended with tris-based extender with no antioxidant (Control), with taurine (40 mM), quercetin (5 μg/ml) and reduced glutathione (5 mM). The post thaw sperm quality was determined by percent sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa and lipid peroxidation was measured in terms of malondialdehyde (MDA) level both in seminal plasma and sperm cell. At post thaw, percent sperm motility and live sperm count were significantly (p < 0.05) higher for taurine than control and reduced glutathione but did not differ significantly (p > 0.05) from quercetin. The percent HOST reacted spermatozoa were significantly higher for taurine than control, quercetin and reduced glutathione. Seminal plasma MDA level was significantly (p < 0.05) lower for taurine than control and non-significantly lower than quercetin and reduced glutathione. However, spermatic MDA level did not differ significantly (p > 0.05) among the control and antioxidants. In conclusion, taurine at 40 mM reduced lipid peroxidation and improved post thaw sperm quality of cryopreserved crossbred ram semen. Further, transportation time of semen samples in an ice chest at 4–5 °C may be included as a part of equilibration period, when collection shed and frozen semen unit are located at a distance.     

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen Microscopic and oxidative stress parameters after freeze-thawing process

There is a lack of information regarding lipid peroxidation and antioxidant capacity in cryopreserved ram semen, and cryopreservation is associated with the production of reactive oxygen species (ROS) which lead to lipid peroxidation (LPO) of sperm membranes, resulting in a loss of motility, viability and fertility of sperm. The aim of this study was to determine the influence of certain additives and their different doses on standard semen parameters, lipid peroxidation and antioxidant activities after the cryopreservation/thawing of ram semen. Ejaculates collected from four Akkaraman rams, a native breed of sheep, were evaluated and pooled at 33 degrees C. Semen samples which were diluted with a Tris-based extender containing additives including trehalose (50, 100mM), taurine (25, 50mM), cysteamine (5, 10mM), and hyaluronan (0.5, 1mg/ml), and an extender containing no additives (control) were cooled to 5 degrees C and frozen in 0.25ml French straws, being stored in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. The use of a Tris-based extender supplemented with 50mM trehalose, 25mM taurine, and 5 and 10mM cysteamine led to higher percentages of post-thaw motility, in comparison to the control group (P<0.01). No significant differences were observed in the percentages of acrosome and total abnormalities, and the hypoosmotic swelling test upon the supplementation of the freezing extender with antioxidants after the thawing of semen. In biochemical assays, the addition of antioxidants did not cause significant differences in levels of malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-Px), after thawing, when compared to groups with no additives. In this study, catalase (CAT) activities were higher in the group that was applied 25mM taurine as an antioxidant, than in all of the other groups (P<0.001). Compared to the controls, antioxidant treatment with 100mM trehalose, 50mM taurine, 5mM cysteamine and 0.5mg/ml hyaluronan, significantly elevated vitamin E (vit E) levels in samples (P<0.001).     

Effect of milk replacer and rumen inert fat on growth and reproduction of Malpura ram lambs

The objective of this study was to assess the effects of milk replacer and rumen inert fat on growth, testicular development, puberty, semen production and sperm motion characteristics of ram lambs reared under intensive management in semi-arid climatic conditions. Seven-day-old male lambs of Malpura breed (n=20) were divided equally into two groups. Up to weaning, the lambs in G1 group (control) were fed concentrate, green khejri (Prosopis cineraria) leaves and cowpea (Vigna unguiculata) hay along with suckling of dams, whereas lambs in G2 group were fed reconstituted milk at 17 g/lamb per day for the 1st week and at 34 g/lamb per day from 2nd week in addition to the feed inputs given in G1. During post weaning, lambs in the G1 group were given control concentrate, whereas in G2 the control concentrate supplemented with 40 g rumen inert fat per kg of feed was offered along with dry pala (Zizyphus nummularia) and ardu (Ailanthus excelsa) leaves. BWs of lambs were recorded weekly up to 6 months of age. Ram lambs of both the groups were trained for semen collection at a weekly interval from the age of 5 months and simultaneously testicular measurements were recorded fortnightly. The feeding of milk replacer and rumen inert fat had positive (P<0.05) effects on BW, testicular length, testicular volume, semen volume, sperm concentration, mass motility, % motility, % rapid, medium or slow motile spermatozoa. However, no significant effect was observed on testicular breadth, scrotal circumference, age of puberty, sperm velocities and other CASA-derived parameters. The results of this study indicate that higher plane of nutrition in the form of milk-replacer feeding during preweaning and rumen inert fat-supplemented feed during the postweaning period to growing ram lambs enhances their growth, testicular development and semen quality.     

Relationships between Brucella ovis semen culture and various semen and serology parameters

Semen and blood samples from 154 rams from two Montana range flocks (Flock A, vaccinated for Brucella ovis ; Flock B, nonvaccinated) were evaluated to determine the relationship between Brucella ovis (B. ovis ) semen culture results and various semen and blood parameters. All rams utilized in this study exhibited no palpable ram epididymitis lesions. Thirteen and 25.6% of the rams tested in Flocks A and B, respectively, had positive B. ovis semen cultures. Only age of ram and ram condition scores differed (P<0.05) between flocks. No flock by semen culture interactions were detected (P>0.05) for any of the parameters evaluated. Age of ram, ram condition score, and spermatozoa rate from forward movement were unrelated (P>0.05) to B. ovis culture results. Rams with positive B. ovis semen cultures had lower sperm motility (P<0.05), higher percentage of abnormal spermatozoa cells (P<0.05), higher percentage of spermatozoa head abnormalities (P<0.01), lower percentage of live-normal cells (P<0.05), higher incidence of white blood cells in semen (P<0.01) and higher complement fixation (CF) titers (P<0.01).     

Semen quality, testosterone, seminal plasma biochemical and antioxidant profiles of rabbit bucks fed diets supplemented with different concentrations of soybean lecithin

A total of 28 adult V-line rabbits were fed ad libitum a control diet or a diet supplemented with 0.5%, 1.0% and 1.5% soybean lecithin (SL) for 12 weeks. Bucks that received 0.5%, 1.0% or 1.5% dietary SL had a higher ejaculate volume, mass motility, sperm concentration, total sperm output and total motile sperm. Dietary SL reduced the percentage of dead sperm and increased the normal sperm, and this concurred with an increase in blood testosterone concentration. Blood and seminal plasma total lipid, acid phosphatase and seminal plasma alkaline phosphatase were significantly increased because of inclusion of SL. Interestingly, SL reduced blood and seminal plasma thiobarbituric acid-reactive substances while increasing blood and seminal plasma glutathione content, glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity. Conception rate and litter size at birth and weaning were also significantly improved. Practically, it could be suggested that SL is a suitable supplement for improving semen quality, antioxidant status, reproductive traits and the economic efficiency of V-line rabbit bucks and 1% is an adequate concentration.     

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

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

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.     

Evaluation of the effects of different trypsin treatments on semen quality after BHV-1 inactivation, and a comparison of the results before and after freezing, assessed by a computer image analyzer

Semen infected experimentally with infectious bovine rhinotracheitis virus (BHV-1) was treated with trypsin at concentrations of 0.30%, 0.25% and 0.15%, with or without (w or w/o) trypsin inhibitor in order to render the semen virus free. The trypsin treatments (at 0.30% and 0.25% by concentration) inactivating the virus up to 10(4) TCID50/ml, and its effects on semen quality were assessed weekly from the 1st to 20th week after being frozen. The following parameters were determined using a computerized semen analysis system (Hamilton Thorn motility analyzer, HTM): total motility, progressive motility and linearity of sperm cells. The results showed that the total and progressive motility of sperm cells were reduced in frozen/thawed semen, principally in the semen treated with trypsin at concentrations of 0.30%. Moreover, the plasma membranes were damaged by trypsin treatments (0.30% by concentration), as determined by the hypoosmotic swelling test (HOS test). These findings suggest that trypsin treatments were effective against the virus however the effects on semen quality and the possibility of a decrease in semen fertility were clear. Trypsin treatment could be recommended at a maximum concentration of 0.25% (w/o trypsin inhibitor) on semen with a high concentration and high motility values of spermatozoa before freezing.     

Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric,microscopic and oxidative parameters in ram semen

This investigation was carried out to study the effect of soybean lecithin 1.5% (wt/vol) (0, 2.5, 5 and 7.5 mg l⁻¹ pomegranate extract (PE)) or PE-loaded lecithin nanoliposome (0, 2.5, 5 and 7.5 mg l⁻¹) to Tris-based extender. Sperm motility (CASA), viability, membrane integrity (HOS test), abnormalities, mitochondrial activity, apoptosis status, lipid peroxidation, total antioxidant capacity (TAC)) and antioxidant activities (GPX, SOD) were investigated following freeze-thawing. No significant differences were detected in motility parameters, viability, membrane integrity, and mitochondria activity after thawing sperm between soybean lecithin and lecithin nanoliposomes. It was shown that PE5 significantly improved sperm total and progressive motility, membrane integrity, viability, mitochondria activity, TAC and reduced lipid peroxidation (malondialdehyde concentration). Moreover, the percentage of apoptotic sperm in PE5 extenders was significantly the lowest among other treatments. Sperm abnormalities, SOD and GPX were not affected by the antioxidant supplements. For apoptotic status, no differences were observed between soybean lecithin and lecithin nanoliposome. We showed that lecithin nanoliposome extender can be a beneficial alternative extender to protect ram sperm during cryopreservation without any adverse effects. It was also observed that regarding pomegranate concentration, PE5 can improve the quality of ram semen after thawing.     

The post-thaw quality of ram sperm held for 0 to 48 h at 5 degrees C prior to cryopreservation

The effects of holding diluted ram semen at 5 degrees C for up to 48 h prior to cryopreservation were investigated. Semen from six rams was collected by electro-ejaculation in the autumn and again from six different rams in the spring. The sperm concentration and motility were determined using spectrophotometry and computerized automated semen analysis, respectively. Samples were diluted at 23 degrees C to 400 x 10(6)cells/ml in a one-step Tris-egg yolk-glycerol (5%, v/v) media, cooled to 5 degrees C over 2h and maintained at 5 degrees C for the duration of the experiments. Aliquots were loaded into 0.5 ml French straws at 0, 24 or 48 h after cooling, frozen in liquid nitrogen vapor for 12-13 min, 4.5 cm above the liquid nitrogen, and plunged into liquid nitrogen for storage. After thawing, autumn samples frozen after 0, 24, or 48 h of storage exhibited similar percentages of motility (29, 31, 36%, respectively), progressively motility (16, 15, 17%, respectively), plasma membrane integrity (28, 35, 29%, respectively) and live acrosome-reacted cells (0.4, 0.6, 0.8%, respectively; P>0.05). In addition, the quantity of sperm that bound to hen's egg perivitelline membranes after being held at 5 degrees C for 0, 24, or 48 h was not significantly different when the values were expressed as means of the quantity of sperm (155, 177, 106 sperm, respectively) or as the proportion of sperm inseminated (0.39, 0.49, 0.34, respectively; P>0.05). Likewise, ram sperm collected in the spring and frozen at 0, 24 and 48 h after cooling had similar (P>0.05) total motility (21, 25, 20%, respectively), progressive motility (14, 15, 11%, respectively), plasma membrane integrity (26, 33, 31%, respectively) and live acrosome-reacted cells (3.7, 3.5, 3.2%, respectively; P>0.05). The 0 h holding time had significantly less sperm bound to a hen's egg perivitelline membrane compared to the 48 h holding time (250 and 470 sperm, respectively) although the 24h holding time was not different from the 0 or 48 h holding time (281 sperm; P<0.05) but analysis of the proportion of the total sperm inseminated resulted in no significant differences observed (P>0.05). These results indicate that ram sperm can be held at 5 degrees C for up to 48 h prior to freezing with no injurious effects on motility, membrane integrity, or fertilizing potential as indicated by membrane binding ability.     

Evaluation of seasonal variations of semen freezability in Leccese ram

The experiment was carried out in Southern Italy (41 degrees N latitude) to examine the effects of seasonal variations of semen freezability in Leccese ram. Semen from five rams, collected every 2 weeks for a whole year, was frozen in straws, using a system based on Tris-fructose egg yolk as extender to constitute semen doses of 100x10(6) spermatozoa. Post-thaw survival and acrosomal status of cells were assessed by dual staining by Hoechst 33258 and FITC-PSA. Three different forms of fluorescence distribution were displayed indicating sperm without acrosome (unstained cells), sperm with damaged acrosome (cells with incomplete fluorescence over the head), sperm with widespread fluorescence (cells completely fluorescent). Motility and kinetic rating at thawing and after 1 and 3h incubation (37 degrees C) were also assessed.Semen frozen in summer and autumn, corresponding to the breeding season, showed the highest (P<0.01) post-thaw survival of spermatozoa (41.7%) and the lowest (P<0.01) incidence of spermatozoa with damaged acrosome. The positive influence of the summer-autumn period was expressed also on motility and kinetic rating of spermatozoa at thawing. The integrity of the acrosomal membrane was positively correlated (P<0.01) with sperm viability before processing (r=0.32) and after thawing (r=0.51).In conclusion, the results show that season exerts a significant influence on semen freezability in Leccese ram, with the best performance occurring the summer and autumn period, corresponding to the reproductive season in temperate zones.