Thaw Motility Research Articles

Role of glucose in enhancing life and potency of Cirrhinus mrigala spermatozoa during cryopreservation

Cryopreservation of fish gametes is an emerging technology and breeding with cryopreserved gametes is advancement in fish seed production. Success of cryopreservation is evaluated by the post - thaw motility of the spermatozoa, an for which energy is required. Cryopreservation is known to cause changes in the seminal plasma that would alter the energy supply for the motility of the spermatozoa. Therefore, energy supplementation is found to be useful during cryopreservation. Cirrhinus mrigala spermatozoa were cryopreserved along with glucose as a co-cryoprotectant after 1:100 dilutions with 0.85% physiological saline as extender and Dimethyl Sulfoxide (DMSO) as cryoprotectant (85:15). The diluents contained glucose at four different concentrations, viz., T1 (0.25%), T2 (0.5%), T3 (0.75%) and T4 (1%). The diluted milt was equilibrated for 10 min at 5? C and loaded into 0.25 ml straws. The loaded straws were then frozen with LN2 vapour for 5 min and immersed in liquid nitrogen. Observations were made once in 7 days for 42 days on motility parameters based on which the duration, score, pattern and percentage were determined. The spermatozoa cryopreserved with glucose at 0.5% concentration showed the highest motility duration of 204±3.6 s whereas Control group showed motility duration of only 83.33± 4.5 s on 42nd day. The difference in motility duration was statistically significant (P>0.025).The present study revealed the benefits of adding glucose a t0.5% during cryopreservation as it could help in maintaining the motility duration and survival of spermatozoa.

Screening of dairy breeding bulls for chromosomal profile and its andrological attributes

Reproductive efficiency is a critical component and the most crucial issue faced by the dairy industries in India and abroad. The present study aimed at assessing the occurrences of male spermatological anomalies leading to poor reproductive performance and to study the chromosomal profile (G-banding) in dairy breeding bulls. Hypo-osmotic swelling test (HOST) and in vitro acrosome reaction were studied using fresh ejaculates of dairy breeding bulls. The metaphase plates did not reveal any chromosomal anomaly (either structural or numerical) in both the species. HOST results indicated significant differences between categories (high and low post thaw motility) but the scores did not differ significantly between the species. In vitro acrosome reaction showed that the bovine species showed significant difference (p<0.05); between various incubation periods (viz. 0, 4 and 6 hours) and the categories differed significantly between nonreacted grades spermatozoa (p<0.01). Although, the present study could not identify any chromosomal anomaly, in generally, the breeding bulls maintained for semen production, should be screened for cytogenetic profile and the sperm membrane integrity.

Semen Quality Parameters of Freezable and Non-Freezable Ejaculates of Mithun (Bos frontalis) Bulls

A study was designed to measure the semen quality parameters (SQP) such as volume, colour, concentration, mass activity, percent of sperm motility, viability, total morphological abnor- mality, intactness of acrosome, plasma membrane, nucleus, vanguard distance travelled by sperm in bovine cervical mucus and hydrogen ion concentration in freezable and non-freezable ejaculates of Mithun. Fifty ejaculates were collected from ten matured Mithun bulls and split to freezable and non-freezable ejaculates based on the post thaw motility (40% or more considered as freezable ejac- ulates). The result has shown that SQP differed significantly (P< 0.05) between the freezable and non-freezable ejaculates and freezable ejaculates have significantly (P< 0.05) higher value than the non-freezable ejaculates. However, morphological abnormality and pH were significantly (P< 0.05) higher in non-freezable than in freezable ejaculates. Mass activity was positively (P< 0.05) correlated with motility, liveability, integrity of acrosome, plasma membrane, nucleus and BCMPT and nega- tively correlated (P< 0.05) with morphological abnormality. Concluded that SQPs were significantly higher in freezable than in non-freezable ejaculates and indicates freezable sperm has higher struc- tural stability than the non-freezable sperm caused freezable sperm.

The Effect of Thawing Temperature on Sperm Quality of Friesian Holstein Bulls

The percentage of sperm motility and morphology are important criteria in evaluating the quality of sperm before it is used for artificial insemination (AI). This study was conducted to observe post thawing motility and abnormal morphology of spermatozoa Friesian Holstein (FH). The materials were used 10 straws of FH bulls in the form of 0.25 ml. A total of 10 straws then divided into two treatment groups of thawing in water at 37°C and water 8°C, respectively. An examination of the motility and morphology of spermatozoa abnormalities performed every two hours for two times. Calculating the percentage of sperm motility was done by calculating the percentage of spermatozoa moving forward in the field of view under a microscope with a magnification of 10x. the percentage of abnormal spermatozoa was assessed by William's stain. Spermatozoa morphology was observed by using a microscope magnification of 100x. Abnormalities of spermatozoa were calculated from a total of 200 spermatozoa, either normal or abnormal. At the same thawing time, the motility of FH cattle sperms post thawing in water temperature 37°C had a higher preference than that of post thawing in water temperature 8°C, although it was not significantly different (P &gt; 0.05). Based on morphological aspects, frozen semen used in this study is within the tolerance limit for the total percentage of abnormal sperm morphology between 12% to 23% and normal morphology between 70% to 88%.

Use of chelating agent for optimum post thaw quality of buck semen

Ejaculates (35) from adult Sirohi bucks (2–4 years old) were utilized for the present study to find out the freezability of buck semen at different levels of chelating agent used (ethylene diamine tetra acetic acid - EDTA: 0, 0.01, 0.05 and 0.1%) by conventional method of freezing. The ejaculates were collected twice at weekly intervals using artificial vagina and were extended to maintain sperms concentration approximately 100 million / dose (0.25 ml) with tris- citric acid- fructose (TCF) diluent having 10% (v/v) egg yolk and 6% (v/v) glycerol as cryo protecting agent. Filling and sealing of straws were done at 5ºC in cold handing cabinet after 4 h of equilibration period then straws were vapor frozen for 10 min above 2 cm of liquid nitrogen and finally put in to liquid nitrogen. Post thaw motility, live sperm count, abnormalities, acrosomal integrity and hypo osmotic swelling test had been conducted to know freezability. Analysis of data using SPSS 16 revealed that post thaw motility, live sperm count, abnormalities, acrosomal integrity and hypo osmotic swelling positive spermatozoa differed significantly at different levels of EDTA. The post thaw motility, live sperm count, acrosomal integrity and hypo osmotic swelling positive spermatozoa were significantly highest in 0.1% of EDTA used in the present study. So, 0.01% EDTA can be used as an additive in semen dilutor in routine freezing process for better post thaw recovery of buck semen.

Characteristics and freezability of Tharparkar bull semen

The study was undertaken to know the characteristics of various attributes of ejaculates collected from 6 elite pure Tharparkar bulls using artificial vagina in fresh and cryopreserved semen. The mean values for fresh seminal parameters, viz. seminal volume (ml), mass motility (0–5 scale), sperm concentration (millions/ml), individual motility (%), live sperm (%), intact acrosome (%), total sperm abnormalities (%), hypo osmotic swelling test (HOST -%) and cervical mucus penetration test (CMPT – mm) were 4.40 ± 0.24, 3.15 ± 0.12, 995.16 ± 70.99, 71.88 ± 0.99, 67.23± 1.08, 79.65 ± 1.21, 19.87± 0.54, 60.15 ± 1.76 and 31.56 ± 0.96, respectively. Semen volume and sperm concentration significantly differed between bulls within breed. The semen was cryopreserved after extension maintaining 20 million sperm/ straw, filling and sealing in 0.25 ml straw using programmable bio- freezer (IMV). Cryopreserved semen was assessed after 24 h freezing after thawing. Freezing significantly lowered motility (71.88 ± 0.99 vs 51.16 ± 1.08), intact acrosome (79.65 ± 1.21 vs 72.43 ± 1.42), HOST (60.15 ±1.76 vs 55.81 ±1.72) and CMPT (31.56 ± 0.96 vs 26.90 ± 0.91). Significantly higher percentages of sperm abnormalities (19.87 ± 0.54 vs 23.50 ± 0.82) were observed after freezing. Freezing resulted in significant decline in post thaw motility, acrosomal integrity, HOST and CMPT and increase in sperm abnormalities in Tharparkar bull semen.

English

In this study, motion characteristics of spermatozoa was assessed by computer assisted semen analyser (CASA) for evaluating fertility potential of Sahiwal bulls. Twelve bulls were selected and grouped into two on the basis of age (AGI &lt; 50 months old; AGII &gt; 50 months old) and scrotal circumferences (SCI &lt; 33 cm; SCII &gt; 33 cm). The following CASA parameters i.e., velocity average path (VAP, &mu;m/s), velocity straight line (VSL, &mu;m/s), velocity curvilinear (VCL, &mu;m/s), amplitude of lateral head displacement (ALH, &mu;m), motility (%)(the percentage motile cells of the total) and straightness (STR) were recorded. Results of the study revealed that there is no significant different (p&gt;0.05) in progressive motility either in age or Sc groups of bulls. However, significantly (p&lt;0.05) higher mean post thaw motility was observed after 24 h cryopreservation for the younger (76.40&plusmn;3.07) than the older (65.00&plusmn;3.50) bulls and for larger SC than smaller SC bulls (65.56&plusmn;3.78 vs. 56.56&plusmn;3.78, p&lt;0.05). Similar trends observed at 0 h after freezing were not significantly different (p&gt;0.05) for both age and SC groups. In most motion characteristics especially in motility and linearity of the motion, younger bulls and bulls with larger SC performed better than older bulls and bulls with smaller SC indicating the possibility of selecting bulls at an early age on the basis of testis size to save the money, space and time which otherwise spent on rearing such inferior bulls. This study also clearly indicated that CASA is a good supplementation to aid for selection of breeding bulls. &nbsp; Key words: Sahiwal bull, computer-assisted semen analysis (CASA) parameters, spermatozoa motion characteristics.

The effect of Equex STM® in freezing media on post thaw motility, viability and DNA integrity of frozen-thawed ram spermatozoa.

In this study we investigated the effect of Equex STM® on quality and in-vitro survival of ram spermatozoa frozen in Tris egg yolk based extender. Ejaculates from 6 crossbreed rams were frozen according to the standard procedure after two step dilution with Tris-egg yolk extender (1). The second extender, added to the semen at 5°C, contained 14% of glycerol and was supplemented with detergent 0.75% Equex STM® (group OEP) or contained no detergent (control group). After thawing the samples were incubated in a water bath at 37°C and analysis were performed 10 minutes, 6, 12 and 24 hours later. Motility and the viability (Viadent®) of the semen were analysed with Hamilton Thorne Biosciences, Version 12.3 and membrane integrity with HOS (hypoosmotic swelling test). DNA fragmentation (DFI %) of F/T spermatozoa was analyzed 10 minutes and 3 hours after thawing using sperm chromatin structure assay (SCSATM). The sperm membrane integrity was analysed 15 minutes and 3 hours after thawing by Sybr-14/PI test. Percentage of motile spermatozoa was significantly higher in OEP group in comparison to control group at 0, 6, 12 and 24 h (P<0.001). Viability of spermatozoa was significantly higher (P<0.001) in OEP compared to control group in all analysed times after thawing. Percentage of HOS positive spermatozoa was significantly higher in OEP compared to control group respectively for 0 (P=0.001), 6 (P=<0.001), 12 and 24 h (P=0.002) after thawing.

Effects of different doses of trehalose supplementation in egg yolk extender in frozen–thawed Angora buck semen

Few studies have been done on the effects of trehalose supplementation in the cryopreservation of Angora buck semen. The objective of present study was to investigate the effects of the addition of trehalose at different doses in semen extenders, on in vitro semen quality parameters, anti-oxidant enzymes activities and DNA damage after the freeze–thaw process in Angora buck semen. Semen samples from 5 mature Angora bucks (3 and 4 years of age) were used in this study. The bucks, belonging to the Livestock Central Research Institute were maintained under uniform breeding condition. A total number of 40 ejaculates were collected twice a week from the bucks using an artificial vagina, during the breeding season and the semen pooled to minimize individual variation. Each pooled ejaculate was split into 7 equal aliquots and diluted (37°C) with base extenders supplemented with the trehalose (12.5, 25, 50, 75, 100 and 150mM), and a base extender with no additives (control). Diluted samples were aspirated into 0.25ml French straws, and equilibrated at 5°C for 4h and then were frozen at a digital freezing machine. The freezing extender supplemented with 50mM trehalose led to the greatest percentages of CASA motility (53.6±4.69), in comparison to the other groups after the freeze–thawing process (P<0.05). The addition of different doses of trehalose did not provide any significant effect on the percentages of post-thaw sperm motion characteristics (VAP, VSL and LIN), compared to the control (P>0.05). The freezing extender with 150mM trehalose group led to the highest percentages of acrosome abnormalities (P<0.05) and 50mM trehalose group had the lowest percentages of total abnormalities (P<0.001), in comparison to the others. There were no significance differences in the DNA integrity among treatment groups (P>0.05). The different doses of trehalose did not show any effectiveness on the maintenance GPx, LPO, GSH, CAT and total antioxidant activities, when compared to the control (P>0.05). Therefore, the additions of 50mM and 75mM doses of trehalose will be useful in increasing post thaw motility on Angora buck semen.

Cryopreservation of Carp Spermatozoa

A successful cryopreservation method would offer many advantages to an aquaculturist. Cryopreservation can provide a year round supply of seeds from desired species regardless of the spawning season and allow hybridization, greater ease in carrying out selective breeding and stock improvement. Studies were conducted on cryopreservation of spermatozoa from Indian major carps. Effect of different concentration of glycerol and equilibration periods on the post thaw motility of spermatozoa from Catla, Rohu and Mrigal were observed. The maximum motility (80 – 85%) was observed with the equilibration period of 20 – 40 minutes with the concentration of 10-15% of glycerol. Rohu showed the same trend with the maximum motility of 78-87%. In Mrigal maximum motility (85-88%) was observed with the equilibration time of (20-40 minutes) with the concentration of glycerol in between (10-15%). Like glycerol, the same type of experiment was conducted with Dimethyl sulphoxide (DMSO). Post thaw motility of cryopreserved spermatozoa in DMSO showed the same way as glycerol. Apart from the above, percentage of motility of spermatozoa was observed with respect to different thawing temperatures. Maximum motility was observed at the thawing temperature of 40o C in catla (85%), Rohu (80.00%) and Mrigal (83%). Interesting results were obtained from the fertilization studies of cryopreserved spermatozoa of Catla with the eggs from the matured Catla breeders. Fertilization rate of 75-80% and the hatching rate of 60% was observed from the above study. From the above results, it could be possible to use cryopreserved spermatozoa for commercial production of fish seeds in fish hatcheries.Keywords: Cryopreservation, Catla, Rohu, Mirgal, DMSO, Fertilization Rate.

Sperm cyropreservation and oxidative damage. What does it mean?

Sperm cyropreservation and oxidative damage. What does it mean?

Effect Of Pre-freeze Addition Of Cysteine Hydrochloride And Reduced Glutathione On Post-thaw Sperm Parameters And Field Fertility In Jersey Crossbred Bull Semen

AbstractThe present study was aimed to assess the effect of pre-freeze addition of cysteine hydrochloride and glutathione (GSH) on post-thaw sperm functional parameters and field fertility. The experimental bulls aged 4 to 6 years were used for the present study. A total of 36 ejaculates, 6 ejaculates from each bull (n=6) were collected and divided in to three groups, group I (control), group II (5mM cysteine hydrochloride) and group III (5mM GSH). The extended semen samples were added with @5mM additives, filled in mini straw using automatic filling and sealing machine (IMV, France) and cryopreserved in liquid nitrogen. Post-freeze seminal traits were also recorded after thawing at 37oC for 30 seconds. Post thaw livability was significantly (p&lt; 0.01) higher in GSH group as compared to cysteine and control groups. The loss of acrosomal integrity was significantly (p&lt; 0.01) lower in GSH group than cysteine and control groups. Analysis of variance for post thaw motility parameters (Forward progressive and Total motility) has revealed that significant difference (p&lt; 0.05) between the good and poor freezer in the group II and there was no difference in the group I and III under study at 0 and 1 hr incubation period and at 2 hrs the group III and at 4 hrs group I has revealed significance difference (p&lt; 0.05).The curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) values were significantly (P&lt;0.05) higher in GSH than the cysteine and control groups. The Mitochondrial membrane potential (per cent) had no significance difference between the treatment groups. The present study indicates that the addition of cysteine and GSH suggestive of reducing lipid peroxide levels. The conception rate (%) in glutathione (68) added semen was significantly (p&gt;0.05) higher than cysteine (58) and control (49) groups. The post-thaw sperm progressive forward motility (r=0.4) had moderate but, no significant correlation with conception rate. However post-thaw VSL (r=0.7), loss of acrosomal integrity (r=-0.8) and mitochondrial membrane potential (r=0.9) had significant (p&lt; 0.05) correlation with field fertility. The present study indicates that the use of glutathione as semen additives may be recommended for improving semen quality and overall augmentation of pregnancy in cows. The present study suggests that pre-freeze addition of glutathione was found to be better than cysteine in improving sperm fertility.

Effect Of Pre-freeze Addition Of Cysteine Hydrochloride And Reduced Glutathione On Post-thaw Sperm Parameters And Field Fertility In Jersey Crossbred Bull Semen

The present study was aimed to assess the effect of pre-freeze addition of cysteine hydrochloride and glutathione (GSH) on post-thaw sperm functional parameters and field fertility. The experimental bulls aged 4 to 6 years were used for the present study. A total of 36 ejaculates, 6 ejaculates from each bull (n=6) were collected and divided in to three groups, group I (control), group II (5mM cysteine hydrochloride) and group III (5mM GSH). The extended semen samples were added with @5mM additives, filled in mini straw using automatic filling and sealing machine (IMV, France) and cryopreserved in liquid nitrogen. Post-freeze seminal traits were also recorded after thawing at 37oC for 30 seconds. Post thaw livability was significantly (p< 0.01) higher in GSH group as compared to cysteine and control groups. The loss of acrosomal integrity was significantly (p< 0.01) lower in GSH group than cysteine and control groups. Analysis of variance for post thaw motility parameters (Forward progressive and Total motility) has revealed that significant difference (p< 0.05) between the good and poor freezer in the group II and there was no difference in the group I and III under study at 0 and 1 hr incubation period and at 2 hrs the group III and at 4 hrs group I has revealed significance difference (p< 0.05).The curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) values were significantly (P<0.05) higher in GSH than the cysteine and control groups. The Mitochondrial membrane potential (per cent) had no significance difference between the treatment groups. The present study indicates that the addition of cysteine and GSH suggestive of reducing lipid peroxide levels. The conception rate (%) in glutathione (68) added semen was significantly (p>0.05) higher than cysteine (58) and control (49) groups. The post-thaw sperm progressive forward motility (r=0.4) had moderate but, no significant correlation with conception rate. However post-thaw VSL (r=0.7), loss of acrosomal integrity (r=-0.8) and mitochondrial membrane potential (r=0.9) had significant (p< 0.05) correlation with field fertility. The present study indicates that the use of glutathione as semen additives may be recommended for improving semen quality and overall augmentation of pregnancy in cows. The present study suggests that pre-freeze addition of glutathione was found to be better than cysteine in improving sperm fertility.

Effect of Seasons on Semen Production Performance of Jersey Bulls

A total of 2084 semen ejaculates collected from 16 Jersey bulls (aged between 2 to 6 years) during three seasons (winter, summer and rainy) of the year were studied to know the seasonal influence on their semen production potential. Overall mean values for various seminal parameters viz. number of ejaculates per month, semen volume (ml), sperm concentration (106/ ml), sperm output per ejaculate (million), initial motility (%) and post thaw motility (%) were 10.85, 4.39 ± 0.04, 944.47 ± 10.07, 4076.27 ± 50.23, 68.20 ± 0.18 and 50.52 ± 0.06, respectively. The season had significant (P<0.05) effect on sperm concentration, sperm output per ejaculate and initial motility; whereas, ejaculate volume and post thaw motility were non-significant. Overall mean values for number of ejaculates per month per bull, sperm output per ejaculate, initial motility and post thaw motility were best observed during the winter season.

Novel animal protein-free sperm cryopreservation media results in post-thaw survival equivalent to standard sperm freezing media

ObjectiveOptimal sperm survival following cryopreservation and thawing is essential, especially in patients with low sperm counts and donor samples. Sperm cryopreservation media has historically been supplemented with proteins sourced from human and/or animal. This study tested a novel media free of human and animal proteins eliminating potential sources of contamination while maintaining cryoprotective properties.DesignProspective comparison of sperm cryopreservation mediaMaterials and Methods15 sperm samples designated for disposal were pooled and washed in Quinn's Fertilization media (Sage) then split for 5 runs with two treatments: standard Freezing media (Test Yolk Buffer, Irvine) or the experimental Cryolink Freezing media (Biogenetics). Freeze media was added drop-wise and gently mixed for 10 minutes. Samples were placed in cryotubes (1ml per tube) and plunged into LN2. On a later date, samples were thawed in a 37° water bath for 5 minutes, mixed with wash media drop-wise over 6 minutes. Samples were washed, pelleted and resuspended in 0.5ml of wash media. Recovery and survival was assessed by count, motility, progression and morphology post thaw and at 0, 3, and 24 hrs after washing. Data was analyzed by Wilcoxen ranked signs test.ResultsTabled 1CRYO-PROTECTANTPRE-FREEZE MOTILITYPOST THAW MOTILITY (0 hr)CELLS RECOVERED (0 hr)POST THAW MOTILITY (3 hr)CELLS RECOVERED (3 hr)POST THAW MOTILITY (24 hr)CELLS RECOVERED (24 hr)TEST YOLK63%45%71%32%51%15%24%CRYOLINK59%47%80%31%63%17%29% Open table in a new tab ConclusionAnimal and/or human proteins are potential sources of bacterial, viral or prion contamination. We tested a novel sperm cryopreservation media prepared free of animal or human protein. No difference in sperm survival post-thaw was observed between Test Yolk buffer and Cryolink media. With further validation, Cryolink media may provide a safer and simplified means of cryopreserving sperm for reproductive use. ObjectiveOptimal sperm survival following cryopreservation and thawing is essential, especially in patients with low sperm counts and donor samples. Sperm cryopreservation media has historically been supplemented with proteins sourced from human and/or animal. This study tested a novel media free of human and animal proteins eliminating potential sources of contamination while maintaining cryoprotective properties. Optimal sperm survival following cryopreservation and thawing is essential, especially in patients with low sperm counts and donor samples. Sperm cryopreservation media has historically been supplemented with proteins sourced from human and/or animal. This study tested a novel media free of human and animal proteins eliminating potential sources of contamination while maintaining cryoprotective properties. DesignProspective comparison of sperm cryopreservation media Prospective comparison of sperm cryopreservation media Materials and Methods15 sperm samples designated for disposal were pooled and washed in Quinn's Fertilization media (Sage) then split for 5 runs with two treatments: standard Freezing media (Test Yolk Buffer, Irvine) or the experimental Cryolink Freezing media (Biogenetics). Freeze media was added drop-wise and gently mixed for 10 minutes. Samples were placed in cryotubes (1ml per tube) and plunged into LN2. On a later date, samples were thawed in a 37° water bath for 5 minutes, mixed with wash media drop-wise over 6 minutes. Samples were washed, pelleted and resuspended in 0.5ml of wash media. Recovery and survival was assessed by count, motility, progression and morphology post thaw and at 0, 3, and 24 hrs after washing. Data was analyzed by Wilcoxen ranked signs test. 15 sperm samples designated for disposal were pooled and washed in Quinn's Fertilization media (Sage) then split for 5 runs with two treatments: standard Freezing media (Test Yolk Buffer, Irvine) or the experimental Cryolink Freezing media (Biogenetics). Freeze media was added drop-wise and gently mixed for 10 minutes. Samples were placed in cryotubes (1ml per tube) and plunged into LN2. On a later date, samples were thawed in a 37° water bath for 5 minutes, mixed with wash media drop-wise over 6 minutes. Samples were washed, pelleted and resuspended in 0.5ml of wash media. Recovery and survival was assessed by count, motility, progression and morphology post thaw and at 0, 3, and 24 hrs after washing. Data was analyzed by Wilcoxen ranked signs test. ResultsTabled 1CRYO-PROTECTANTPRE-FREEZE MOTILITYPOST THAW MOTILITY (0 hr)CELLS RECOVERED (0 hr)POST THAW MOTILITY (3 hr)CELLS RECOVERED (3 hr)POST THAW MOTILITY (24 hr)CELLS RECOVERED (24 hr)TEST YOLK63%45%71%32%51%15%24%CRYOLINK59%47%80%31%63%17%29% Open table in a new tab ConclusionAnimal and/or human proteins are potential sources of bacterial, viral or prion contamination. We tested a novel sperm cryopreservation media prepared free of animal or human protein. No difference in sperm survival post-thaw was observed between Test Yolk buffer and Cryolink media. With further validation, Cryolink media may provide a safer and simplified means of cryopreserving sperm for reproductive use. Animal and/or human proteins are potential sources of bacterial, viral or prion contamination. We tested a novel sperm cryopreservation media prepared free of animal or human protein. No difference in sperm survival post-thaw was observed between Test Yolk buffer and Cryolink media. With further validation, Cryolink media may provide a safer and simplified means of cryopreserving sperm for reproductive use.

The Effect of Centrifugation on Semen Quality of Peranakan Ettawah Goat's Post Thawing

The aim of the research was to determine the optimum of centrifugation on the quality of Peranakan Ettawah goat's spermatozoa prepared for in vitro fertilization preparation.The research of material was frozen semen ofPeranakan Ettawah goat produced by Artificial Insemination Center in Singosari with minimum post thawing motility in 40%. The method of the research was an experiment method with the four treatment, i.e: 1000 (P1), 1500 (P2), 2000 rpm (P3) of different centrifugation. The variable observes were spermatozoa motility, spermatozoa viability, and abnormal morphology of spermatozoa. Data obtain was analyzed statistically using Completely Randomize Design and continued with Tukey test. The result showed that on the upper layer of each treatment (P1, P2, and P3) obtained 55,72±3,55; 69,55±3,35; and 55,19±2,72% for spermatozoa motility, 62,99±5,87; 73,99±4,36; and 57,39±9,22% for spermatozoa viability, and 7,19±3,64; 8,84±2,65; and 6,40±3,00% for abnormal morphology of spermatozoa, descriptively. While on the lower layer the result showed 55,59±3,99; 68,63±3,88; and57,61±2,20% for spermatozoa motility, 70,05±7,47; 77,44±1,08; and 69,93±11,98% for spermatozoa viability, and 10,36±6,20; 9,55±3,27; and 8,09±2,80% for abnormal morphology of spermatozoa, desciptively. It was concluded that centrifugation in 1500 rpm showed the highest motility and viability on the upper layer and lower layer.

Sperm motile efficiency (SME) of fresh spermatozoa in a hypotonic medium correlates with their cryosurvival in fertile men and infertility patients (predicting test).

The sperm motile efficiency (SME) of fresh spermatozoa in a hypotonic medium was investigated concerning the prediction of post thaw motility and the differentiation between fertile men and infertility patients. A total of 95 semen samples were collected for this study. Twenty of these samples were obtained from semen donors of proven fertility and 75 samples were collected from male partners of infertile couples. There were no significant differences between the SME after mixture of the fresh semen with a 140 mOsm saline in equivalent volumes and after cryopreservation (Wilcoxon-matched pairs signed ranks-test) but a significant positive correlation between the SME after both treatments of semen samples was notable. Significant differences between the fertile control group and group of infertility patients could be determined concerning the SME after mixing of fresh semen with 140 mOsm saline as well as after cryopreservation (Mann-Whitney-U-test). The discriminant analysis revealed a most discriminating effectiveness for SME immediately after the addition of hypoosmotic saline.

Freezing sperm from cauda epididymis of castrated stallions

The goal of this work was to compare cryoresistance of epididymal spermatozoa with that of sperm collected with an artificial vagina. For this study, semen was collected from ten stallions using an artificial vagina. After that the stallions were bilaterally castrated and both epididymes dissected. The epididymides were maintained at ambient temperature until sperm recovery from the epididymal tails as described by Granemann et al. (2006). Three groups were established: (1) semen collected with artificial vagina prior to castration, (2) semen collected from the left epididymis, and (3) semen collected from the right epididymis. Spermatozoa were diluted in Botu-Crio® extender to a concentration of 400× 106 sperm/ml and stored in 0.5-ml straws. After 20 min at 5 ◦C, the straws were placed in nitrogen vapour (approximately −120 ◦C) for 15 min, and then plunged into liquid nitrogen (−196 ◦C). The straws were thawed in water bath at 37 ◦C for 30 s. Total and progressive motility and acrosome alterations were evaluated before freezing and after thawing by light microscopy. For the evaluation of acrosomal integrity, the sperm smears were stained with Cerovsky stain (Cerovsky, 1976). Total and progressive motility before freezing were higher (p< 0.05) in group 1 (73 and 58%) than in group 2 (45.7 and 32%) or in group 3 (38.8 and 21.9%). Acrosomal defects did not differ among the groups: 7.6, 10.4, and 7.1% for groups 1, 2, and 3, respectively (p< 0.05). After thawing, total and progressive motility and acrosome defects were similar in all groups: 38.5, 27.7% and 21.7, 25.5% and 18, 12.2% for groups 1, 2 and 3, respectively. Progressive motility of fresh collected spermatozoa was higher in all groups, when compared with progressive motility of epididymal sperm after thawing (p< 0.05). It was possible to preserve only <20% of the epididymal sperm. Recovery techniques for epididymal stallion sperm need to be improved to increase the efficiency of cryopreservation of epididymal sperm, similarly to what has been demonstrated in the post-mortem collection of bovine epididymal sperm (Herrick et al., 2004).

TRIALS TO IMPROVE THE VIABILITY OF RABBIT SEMEN AT VARIABLE CONDITIONS OF PRESERVATION USING DIFFERENT DILUENTS

Ten sexually mature rabbit bucks of (NZW) were used in this study. The present study aimed to investigate the effect of some diluents and differents regimes of dilution and preservation on the viability of rabbit semen . Semen was collected by an artificial vagina and evaluated for volume (ml), individual motility (%); concentration of spermatozoa (ml × 106); percentage of alive spermatozoa and total abnormalities by conventional technique. Pooled semen sample was divided into three parts, the first part was extended in Tris – egg yolk (pH 6.9), the second part was extended in Tris-egg yolk (pH 7.5) and the third part was extended in sodium chloride (pH 7.0). Each part again was subdivided into three portions; the first portion was incubated at 37˚C. for 3 hr.; The second portion was kept at 5 oC. for 48 hr. and the third portion was frozen in French straws (0.25 ml.) and stored in liquid nitrogen ( -196˚C). Our results revealed that the percentage of alive spermatozoa displayed a significant increase (P<0.05) at 37 oC in sodium chloride extender than the other extenders, while there were no significant differences in individual sperm motility in the three extenders at 37 oC. .On the other hand the individual sperm motility signed a significant increase (p < 0.05) in Tris-egg yolk extender with pH 6.9 than the other extenders at 5˚C. The post thawing motility was significantly higher (P<0.05) in Tris-egg yolk extended semen with pH 6.9 than the other extended semen. The extended semen with sodium chloride reported a highly significant percentage in acrosomal defects than the other extended semen with different pH.

RESEARCHES CONCERNING THE QUALITY OF THAWED STALLION SEMEN

During the 2007th breeding season, ten frozen semen samples (1-6 from a stallion from SD Jucu stable and 7-10 from the Centre Haras Nationaux, France) were took in study. The semen was collected using phantom mare and CSU type artifficial vagina. Fresh semen was evaluated by macro and microscopic techics, and the criopreservation was made in the Cluj-Napoca Laboratory of Andrology and Reproduction Biotechnologies. It was performed fast thawing, appreciating the mobility, viability, with/without DIEE Ancu Dinca devices, concentration and morphological examination. The sperm post thawing motility was between 15% and 65% and the viability varied between 22% and 85%, being superior to the motility value. The DIEE device utilization had not a visible influence on post thawing sperm motility and viability. The concentration values, were between 53 mil/ml. and 105 mil/ml .The highest percentage of normal sperm, determined by Spermarc staining, was recorded in the case samples 7 and 8, with 90%, respectively 86%. The samples from Les Breviaires France sperm stocking centre, had a higher motility and viability percentage (65% motility and 85% viability), compare with the ones processed in our laboratory (55% motility and 75% viability). From the 6 ejaculates frozen at USAMV Cluj, 3 samples were considered to be good for insemination