Post Thaw Research Articles

Cryopreservation in rhinoceros—Setting a new benchmark for sperm cryosurvival

At times when rhinoceros are fiercely poached, when some rhinoceros species are closer than ever to extinction, and when the scientific community is in debate over the use of advanced cell technologies as a remaining resort it is time to simplify and improve existing assisted reproduction techniques to enhance breeding and genetic diversity in the living populations under our care. Semen cryopreservation has been performed in all captive rhinoceros species with limited degree of success. Here we tested three freezing extenders, containing different cryoprotectants and various freezing rates for the cryopreservation of rhinoceros sperm from 14 bulls. In experiment I, semen from 9 bulls was used to determine the most suitable diluent, cryoprotectant and freezing rate for the successful cryopreservation of rhinoceros sperm. In experiment II, semen from 5 bulls was used to assess whether the removal of seminal plasma could further improve post thaw sperm quality following cryopreservation with conditions identified in Experiment I. Semen was diluted with Berliner Cryomedia, ButoCrio® or INRA Freeze®, packaged in 0.5 mL straws and frozen 3, 4, and 5 cm over liquid nitrogen (LN) vapour or directly in a dryshipper. It was found that semen extended with ButoCrio® (containing glycerol and methylformamide) and frozen 3cm over LN vapour provided the best protection to rhinoceros spermatozoa during cryopreservation. When pooled over treatments, total and progressive post thaw motility was 75.3 ± 4.2% and 68.5 ± 5.7%, respectively marking a new benchmark for the cryopreservation of rhinoceros sperm. Post thaw total and progressive motility, viability and acrosome integrity of semen diluted in ButoCrio® was significantly higher than semen extended in Berliner Cryomedia or INRA Freeze®. The removal of seminal plasma did not improve post thaw sperm survival (p > 0.05). In conclusion, the cryosurvival of rhinoceros spermatozoa was significantly improved when using a mixture of glycerol and methylformamide in combination with a fast freezing rate at 3 cm. These results describe a new protocol for the improved cryosurvival of rhinoceros spermatozoa and will enable a more successful preservation of genetic diversity between males, especially in donors whose spermatozoa may already be compromised prior to or during collection. The successful reduction of glycerol concentration in favour of methylformamide as a cryoprotectant could be a novel suggestion for the improvement of cryopreservation techniques in other wildlife species.

Evaluation of pigeon egg yolk for post thaw quality, enzyme leakage and fertility of buffalo (Bubalus bubalis) bull spermatozoa

Egg yolk is used as a cryoprotectant in semen preservation. However, its composition varies according to the species which may influence its effectiveness during the freeze-thaw process. Therefore, study was conducted to identify the optimum level of pigeon egg yolk (PEY) in Tris citric acid (TCA) extender for freezability and in vivo fertility of buffalo semen. Semen was collected at weekly intervals for a period of three weeks (replicates) from 6 Nili Ravi buffalo bulls (2 ejaculates/bull/replicate) and diluted with TCA extender (50 × 106 motile spermatozoa ml−1) containing 5%, 10%, 15% and 20% PEY or 20% CEY (control) and cryopreserved. Post-thaw sperm quality and extracellular enzymes leakage was assessed after thawing. Sperm motility, plasma membrane integrity, livability and viability was significantly higher in extenders containing 10% and 15% PEY compared to 5% PEY, 20% PEY or 20% CEY (controls). A dose-dependent decrease was recorded in the chromatin damage for the PEY, being lowest for the 15% and 20% PEY which was significantly less compared to controls (20% CEY). The extracellular GOT and LDH leakage was significantly lower (P < 0.05) in extender containing 10% and 15% PEY compared to the controls. Semen collected from 2 bulls, cryopreserved in extenders containing 15% PEY or 20% chicken egg yolk was assessed for fertility after artificial inseminations. A total of 400 buffaloes were inseminated (100 inseminations/extender/bull). The overall fertility rate was significantly higher (P < 0.05) with semen cryopreserved in extender containing 15% PEY (56%) compared to 20% CEY (42%; controls). In conclusion, pigeon egg yolk at 15% offers advantages over 20% chicken egg yolk in terms of in vitro post-thaw semen quality and in vivo fertility of buffalo.

Quercetin Promotes Increase in the Fertility Rate of Frozen Semen of Stallions Considered Sensitive to Freezing

Aim of this study was to evaluate the stimulating effects of quercetin (QUE) on post thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa. QUE is a well-known flavonoid having antioxidant properties to scavenge free radicals. Semen was collected from three buffalo bull through artificial vagina (42 °C) and were evaluated initially for volume, motility, concentration, followed by dilution in five extenders (n = 5 aliquots, control, C = no QUE; treatment 1, T1 = 50 μM QUE; treatment 2, T2 = 100 μM QUE; treatment 3, T3 = 150 μM QUE and treatment 4, T4 = 200 μM QUE supplementation). The experiment was repeated thrice (n = 3 replicates). At post dilution, sperm progressive motility (PM, %) plasma membrane integrity (PMI, %), supra vital plasma membrane integrity (SVPMI, %) and acrosome integrity (ACR-1, %) of buffalo bull were significantly higher (P < 0.05) in extenders supplemented with QUE than C. At thawing, sperm PM of buffalo bull was higher in T3 and T4 than C, T1 and T2. Sperm SVPMI was significantly higher in T2, T3 and T4 than C and T1. Sperm ACR-I was higher in T3 and T4 than C, T1 and T2. Sperm DNA integrity was higher in T4 than C. QUE supplementation increased in vitro semen quality during 30 and 60 min of incubation at 37 °C than C. The in vivo fertility was higher in T3 and T4 than C (%, 61.82 vs. 65.22 vs. 46.90). It is concluded that QUE @ 150 and 200 μM improved post thaw semen quality and in vivo fertility of buffalo bull.

Combination of l-cystein and reduced glutathione in tris-citric acid based extender improves the post thaw quality of dromedary camel spermatozoa

Orexin A belongs to the group of hypothalamic-derived peptides that are involved in a number of processes, such as regulation of energy metabolism, control of food intake and regulation of the reproductive system, by influencing the hypothalamic-pituitary-ovarian axis. Orexin A is also present in the endometrium, myometrium and placenta, which indicates that it may function as an important local regulator of the reproductive functions. The aim of this study was to explore the effects of orexin A on global gene expression in the endometrium of pigs during early gestation, on days 15 to 16 of pregnancy (implantation period). Orexin A altered the expression of 1,242 genes. In this group, 1,104 genes had a fold change greater than 1.2 (P < 0.05). In the group of genes with a fold change that was greater than 1.2, the expression of 457 genes were increased and 647 decreased because of the effects of orexin A. An analysis of the interactions between differentially expressed genes demonstrated that orexin A interacts with genes that potentially encode intracellular signalling pathways and factors regulating reproductive functions in the endometrium. The data from the present study indicate that orexin A affects a number of genes and processes in the endometrium of pregnant pigs and may be regarded as an important regulator of implantation, depending on maternal nutritional status.

Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage

Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.

Impact of l-carnitine on lipid content and post thaw survivability of buffalo embryos produced in vitro

The aim of the present study was to see the impact of L-Carnitine (LC) on lipid biosynthesis and metabolism of buffalo embryos, and post thaw blastocyst survivability. In vitro fertilized (IVF) embryos were derived from slaughterhouse derived COCs and cultured in different doses of LC i.e. 0, 1 mM, 1.5 mM, 2 mM starting at 48 h post IVF. Blastocyst rate was significantly (p < 0.05) higher in 1.5 mM group than control and 1.0 mM group. Lipid content was measured indirectly by fluorescent intensity of lipid droplets after Nile red staining, and it was lower (p < 0.05) in treated than control groups. CPT1B, DGAT2 and DGAT1 mRNA expression was up regulated (p < 0.05) while AMPKg1 expression was down regulated in 1.5 mM and 2 mM groups compared to other groups (p < 0.05). mRNA expression of GLUT1, OCT4 and IFN-tau was higher (P < 0.05) in 1.5 mM group than the control group. Expression of BAX was down regulated at 1.5 mM LC. Blastocyts were vitrified by a modified OPS method and post thaw survivability of blastocysts was higher (P < 0.05) in 1.5 mM LC than other groups. In post thaw blastocysts, mRNA expression of GLUT1, OCT4 and IFN-tau was higher (P < 0.05) in 1.5 mM than other groups. Thus, it can be concluded that supplementation of l-carnitine (1.5 mM) in embryo culture media improved the quality of buffalo embryo production and post thaw blastocysts survivability by reducing fatty acid synthesis, enhancing fatty acid metabolism, and reducing lipid droplet formation.

A comparative study of effects of different freezing methods on sperm quality, DNA integrity and membrane protein of cryopreserved boar semen*

The present study determined the effects of conventional, and controlled freezing method adopting three freezing rates, viz. 20°C, 40°C and 60°C/min on quality of sperm (motility, viability and plasma membrane integrity), DNA integrity and plasma membrane protein profile of cryopreserved boar semen. Sixty sperm-rich fractions of ejaculates from six boars were utilized for freezing of semen with different freezing methods in lactose-egg yolk glycerol extender. Semen samples were evaluated for sperm motility, viability (Propidium Iodide assay), functional integrity of plasma membrane (HOST), DNA integrity (Acridine Orange stain) and plasma membrane protein profile (SDSPAGE) after equilibration and after freezing. The results revealed that the post thaw sperm motility, sperm viability, and plasma membrane integrity (HOST-reacted) were significantly higher in all the three controlled freezing methods (20°C, 40°C and 60°C/min) as compared to that in conventional method. In addition, the number of sperm plasma membrane protein loss was less in controlled freezing methods as compared to that in conventional freezing. However, the post thaw sperm DNA integrity did not influence by difference in freezing methods. No significant difference on the post thaw sperm characteristics was recorded among the three controlled freezing rates. All the sperm parameters assessed declined significantly after freezing as compared to that after equilibration irrespective of freezing method employed. In conclusion, controlled freezing methods conferred better post thaw sperm quality as compared to conventional method, and thus the freezing rates of either 20, 40 or 60°C/min could provide better freezability of boar semen.

Evaluation of semen quality of buffalo frozen semen produced by Artificial Insemination Center

This study aimed to evaluate and compare the quality of post thawing spermatozoa of buffalo frozen semen produced by artificial insemination centers on standard values, and proposed reference values (PRV). Materials of the research were 60 samples of straws obtained from three Artificial Insemination Center, which are each 20 straws, respectively. Parameters observed were motility, concentration, longevity, plasma membrane integrity (PMI), acrosome integrity (AIn) and recovery rate. The obtained data were tested by Z test then presented as mean ± standard deviation. The research results from three artificial insemination center showed that motility mean was 45.00±3.07%, concentration mean was 26.09±3.11 x106 cells/0.25mL, longevity mean was 10.38±0.75%/hour, PMI mean was 45.86±10.67%, AI mean was 73.99±5.28% and recovery rate mean was 64.38±5.16%. The conclusions of this research were the motility and concentration mean were higher than Indonesian National Standard (INS), longevity mean was lower than Department of Animal Husbandry, Dairying and Fisheries (DADF) standard but PMI and AIn mean were higher, the mean value of recovery rate was higher than of proposal value.

Feasibility of cord blood bank in high altitude Abha: preclinical impacts.

We explored the possibility of the cryo-storage of cord blood hematopoietic stem cells (CBHPSC) with respect to the quantity, quality and biologic efficacy of high altitude (HA) region Abha against sea level (SL) region. The results of the post-processed total nucleated cell count was 8.03±0.31×107 and 8.44±0.23×107 cells in the HA and SL regions respectively. The mean post processing viability of the nucleated cells was about 87.03±1.39 (HA) and 88.33±1.55% (SL) while post thaw cells were 85.61±1.44 (HA) and 86.58±1.61% (SL) after transient cryo-storage. The proliferation of CBHSCs after thawing were comparable between the HA and SL regions. The results of the colony forming unit (CFU) assays of CFU-E, CFU-GEMM, CFU-GM and BFU-E were comparable between HA and SL in both fresh and post thaw, while a declining trend with viability was significant. The differentiation capability of post thaw samples into adipocytes and osteocytes were comparable between HA and SL regions. Overall from the results, it can be evidenced that HA cord blood collection, processing or storage does not hinder the quality or biological efficacy of the CBHPSC.

Colloid centrifugation of fresh semen improves post-thaw quality of cryopreserved dromedary camel spermatozoa

Colloids have been successfully used in a number of species to improve sperm populations for IVF and for cryopreservation The usefulness of Single Layer Centrifugation (SLC) for freezing dromedary camel spermatozoa in two different extenders was evaluated by examining the motility, viability, acrosome status, DNA integrity, and ability of cryopreserved sperm to penetrate oocytes in vitro in a heterologus IVF system. Two ejaculates from each of five males were divided into four aliquots: two were processed by SLC (selected) while two were centrifuged without colloid (control). Pellets were cryopreserved in Green Buffer or INRA-96® containing 3% glycerol and evaluated at 0 and 1 h post thawed. The SLC improved post-thaw total and progressive motility at 0 (both P < 0.0001) and 1 (P < 0.001; P < 0.01, respectively) h, and STR (both P < 0.05) and BCF (both P < 0.001) at 0 h. Sperm viability and acrosome integrity (both P < 0.001) were improved at both time points. Sperm frozen in Green Buffer had greater total and progressive motilities at 0 (both P < 0.001) and 1 (both P < 0.001) h than INRA-96® samples. Spermatozoa in Green Buffer also had a greater VAP, VCL and VSL at 0 h and improved viability and acrosome integrity at 0 h (P < 0.05; P = 0.001, respectively) and 1 h (P < 0.05; P < 0.001, respectively). Viability of SLC spermatozoa was improved in Green Buffer at 1 h (P < 0.05). Oocyte penetration (P < 0.05) and pronuclear formation (P < 0.01) were greater with SLC-selected spermatozoa than non-selected spermatozoa, regardless of extender. No difference was observed between treatments or extenders in the mean number of spermatozoa per oocyte penetrated. The SLC spermatozoa had less (P < 0.01) DNA fragmentation compared to controls. The DNA fragmentation was moderately and negatively correlated with penetration (r = −0.4162; P = 0.02) and pronuclear formation (r = −0.3390; P < 0.01). In conclusion, colloid centrifugation of spermatozoa and cryopreservation in Green Buffer improves post thaw motility variables and IVF performance of dromedary camel spermatozoa.

Influence of Seasons and Extenders on Quality and Freezability of Gir Bull Semen under Middle Gujarat Climate

The study was conducted to evaluate the seasonal influence (peak winter and summer) and the efficacy of three extenders (egg yolk based TFYG extender and egg yolk free soya bean based commercial extenders Optixcell and Andromed) on quality and freezability of Gir bull semen in Middle Gujarat. Semen ejaculates (6/bull/season, total36) revealed mean ejaculate volume 6.49±0.30 ml, sperm concentration1212.36±58.10 million/ml, progressive motility 74.17±0.78 %, live sperm 81.39±0.80 %, abnormal sperm 7.36±0.31 %, and sperm with intact plasma membrane 81.31±0.98 % and intact acrosome 94.81±0.24 %. Only the progressive sperm motility was significantly (P&lt;0.05) higher(76.39±0.97 % vs. 71.94±1.00 %) with lesser sperm abnormality(6.17±0.37 % vs. 8.56±0.30 %) during winter than in summer. Semen samples split diluted with TFYG, Optixcell and Andromed extenders recorded the overall mean values of progressive sperm motility, livability, abnormality, plasma membrane integrity and acrosomal integrity during winter season as 77.87±0.51, 77.50±0.45, 5.56±0.20,76.02±0.81 and 94.35±0.29 on dilution; 72.41±0.51, 70.50±0.64, 5.96±0.26, 71.20±0.79 and 93.09±0.32 at pre-freeze stage; 41.30 ±0.94, 50.28±1.03, 9.15±0.31, 29.89±0.40 and 90.65±0.40 at post-thaw stage, respectively. The respective values in summer season were 72.13±0.60, 75.50±0.60, 7.48±0.25, 75.61 ±0.55 and 94.09±0.30 on dilution; 65.46±0.66, 69.41±1.05, 8.89±0.28, 69.70±0.66 and 92.63 ±0.33 at pre-freeze stage; 31.48±0.52, 45.09±0.85, 13.48±0.33, 26.85±0.71 and91.26±0.38 at post-thaw stage. The overall mean sperm post-thaw motility/longevity at 0, 30, 60 and 120 min of incubation at 37°C was 41.20±1.51, 35.19±1.47, 28.80±1.75 and 17.50±1.47 % during winter season and 31.57±0.89, 26.20±0.77, 20.37±0.83 and 13.80±0.77% in summer season, respectively. The initial quality as well as freezability of semen in terms of motile, live, normal and HOS reactive sperm including post thaw longevity were better in winter season than in summer season. Further, the values of all the five semen quality parameters studied were comparatively better in Optixcell than TFYG and Andromed extenders with significant differences only in sperm progressive motility in both the seasons.The season x extender interaction was not significant for any of the sperm quality parameters studied.

Expression and localization of aquaporins 3 and 7 in bull spermatozoa and their relevance to sperm motility after cryopreservation.

Artificial insemination with cryopreserved semen is a well-developed technique commonly used for controlled reproduction in cattle. However, despite current technical advances, cryopreservation continues to damage bull spermatozoa, resulting in a loss of approximately 30 to 50% of viable spermatozoa post thawing. To further improve the efficiency of cryopreservation of bull spermatozoa, understanding the molecular mechanisms underlying the cryobiological properties that affect cryoinjuries during cryopreservation process of bull spermatozoa is required. In this study, we examined the expression and localization of aquaporin (AQP) 3 and AQP7 in fresh, cooled, and frozen-thawed bull spermatozoa. Furthermore, we investigated the relevance of AQP3 and AQP7 to motility and to membrane integrity in frozen-thawed bull spermatozoa. Western blotting against AQP3 and AQP7 in bull spermatozoa revealed bands with molecular weights of approximately 42 kDa and 53 kDa, respectively. In immunocytochemistry analyses, immunostaining of AQP3 was clearly observed in the principal piece of the sperm tail. Two immunostaining patterns were observed for AQP7 ―pattern 1: diffuse staining in head and entire tail, and pattern 2: diffuse staining in head and clear staining in mid-piece. Cooling and freeze-thawing did not affect the localization pattern of AQP7 and the relative abundances of AQP3 and AQP7 evaluated by Western blotting. Furthermore, we demonstrated that the relative abundances of AQP3 and AQP7 varied among ejaculates, and they were positively related to sperm motility, particularly sperm velocity, post freeze-thawing. Our findings suggest that AQP3 and AQP7 are possibly involved in the tolerance to freeze-thawing in bull spermatozoa, particularly in the sperm’s tail.

Improvement in cryosurvival of buffalo bull (Bubalus bubalis) sperm by altering freezing rate within critical temperature range

Objective: To optimize the cryopreservation of buffalo bull semen by altering freezing rates within critical temperature range (4 °C to -60 °C). Methods: A total of 20 ejaculates each from 5 Murrah buffalo bulls were cryopreserved using programmable biofreezer in 2 phases. In the 1st phase, 9 freezing rates were applied at -2, -5, -10, -20, -30, -40, -50, -60 or -4 °C/min (control) from 4 °C to -15 °C ; at -40 °C/min from -15 °C to -60 °C. In the 2nd phase, a fixed freezing rate was applied at -30 °C /min from 4 °C to -15 °C. Six freezing rates were applied at -10, -20, -30, -40 (control), -50 or -60 °C/min from -15 °C to -60 °C. The freezing from -60 °C to -140 °C were fixed at -50 °C/min in both the phases. Post thaw semen quality was assessed in terms of motility, viability, membrane integrity (hypo-osmotic swelling test), sperm abnormalities, and active mitochondria. Data were arc sine transformed and analyzed through one-way analysis of variance using SPSS software. Results: In the 1st phase, percent individual motility, progressive motility and viability were similar among various protocols. Percent hypo-osmotic swelling reactive sperm was higher with freezing at -30 °C/min. In the 2nd phase, percent individual motility, viability and hypo-osmotic swelling reactive sperm was higher with freezing at -50 °C /min. Sperm head abnormalities were lower at -30 °C /min in the 1st phase, but were similar among the protocols of the 2nd phase. Percent active mitochondria were higher at -30 °C /min in the 1st phase and at -50 °C/min in the 2nd phase. Conclusions: The optimum post thaw semen quality of buffalo bull could be obtained by applying freezing rate at -30 °C/min (4 °C to -15 °C) and at -50 °C/min (-15 °C to -140 °C ), followed by plunging of straws in into liquid nitrogen for storage.

Epigenetic status of buffalo fibroblasts treated with sodium butyrate a chromatin remodeling agent.

The somatic cells having higher levels of DNA methylation and reducing it in donor cells before nuclear transfer (NT) by treating them with chemicals, may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Therefore, the present study was planned to understand mechanism of epigenetic changes in donor cells (buffalo fibroblasts) treated with different concentration of sodium butyrate (NaBu)-a chromatin remodeling agent. The cultured fibroblasts purity and lineage were confirmed by fibroblast specific protein and gene markers (Vimentin, Tubulin and Cytokeratin) at different passages using immuno-staining and qPCR respectively. The buffalo fibroblast cells were treated with 1, 3 and 5 mM of NaBu and observations were taken on their morphological changes, population doubling time and cell proliferation after 48 h of treatment. The epigenetic changes were observed using acetylation (H3K9ac) and methylation (H3K27me3) markers expression. The fibroblast cells derived from new born ear tissue started emerging and anchoring to cell culture flasks within 24 h and showed spindle-shaped morphology. The confluent culture was cryopreserved at different time interval. The post thaw culture behavior of the cryopreserved cells was also observed at different time interval and passages. The morphology of NaBu treated cells were changed with increase of dosages of NaBu treatment. The increase population doubling times and decreases the proliferation rate in the dose dependent manner. The NaBu treatment showed that the significantly increased the acetylation (H3K9ac) and methylation (H3K27me3) level over the control. Based on the observations of fibroblast characterization as well as epigenetic modifications of these cells after treatment with NaBu, results suggest that the cells may provide a useful approach for better epigenetic reprogramming in SCNT embryos.

Increased Integrity of Plasma Membrane and Acrosome Cap Spermatozoa Limousin Cattle at Post Thawing in Frozen Media by adding Seawater Extract

This research was determined membrane integrity and acrosome cap of Limousin bull post thawing after adding seawater extract with different concentrations in extender skim milk and egg yolk to increased frozen semen quality. This research used fresh samples of Limousin bull’s semen collected by using artificial vagina, then devided into 4 treatments and 6 replications. The experimental design that used was Complete Random Design. Analysis of the data using Analysis of Variant (ANOVA) one way then proceeds to the Duncan Multiple Range Test to determine significant differences between treatments.The first treatment P0 no seawater extract added as a control. The second treatment P1 was treated with 0.109 µL seawater extract, P2 was treated with 0.426 µL seawater extract, and P3 was treated with 1.09 µL seawater extract. The result showed that determine significant differences between treatments. The post thawing membrane integrity’s result was P0= 22.00 ± 4.28, P1= 22.66 ± 3.61, P2= 25.00 ± 2.75, and P3= 29.00 ± 1.67. The post thawing acrosome cap’s result was P0= 30.50 ± 1.37, P1= 31.50 ± 3.27, P2= 34.83 ± 2.31, and P3= 38.00 ± 1.41. The highest concentration added seawater extract to increased membrane integrity and acrosome cap spermatozoa in this research was 1.09 µL. Keywords: Limousin bull; spermatozoa; seawater extract; membrane integrity; acrosome cap

Post Thawing Sperm Quality and Ca+2 Intensity Characters of Local Goat Sperm After Freezing by Simple Method Using Deep Freezing

The objective of this research was to determine the effect of the simple modified freezing method, 1 ° C /minute freezing rate with different diluter ration on a post-thawing quality of local goat sperm namely Peranakan Etawah (PE). This work is aimed to study the quality of post-thawing sperm and to characterize the calcium intensity profile of both fresh and post thawing goat sperm. The method used is the experimental design of a laboratory. Freezing semen was performed in 2 main temperatures of -45 ° C then -196 ° C respectively using Mr. Frosty (®) System. Early Sperm characters of Ca +2 intensity was performed by Confocal Laser Scanning Microscope (CLSM) through Fluo -3 staining and Ca ++ intensity was analysis descriptively. The result showed that post-thawing qualities are considered as good as standard qualities, at least, more than 40% based on Indonesian National Standard (SNI, 2014). The different level diluents commercial of Andromeda used were influenced highly significant (P<0.01). The best diluents ration is 1:4 (v/v) for final sperms stocked at -196 ° C . However freezing sperm conserved in -196 ° C is better than in -45 ° C . Meanwhile, the sperm characters of two condition showed the important variation of Ca +2 intensity, with the length of region measurement of 39.06±4.595 and 32.696±9.011 µm each. It was concluded that the calcium intensity pattern was varied more and higher in fresh sperm than in freezing sperms. This simple modified method of a freezing system was considered as a feasible alternative method for goat semen in a reason for both for sperm post-thawing quality and practical purposes.

Cryoprotective effect of low-density lipoproteins on post thaw semen quality in Hariana bull

The study aimed to evaluate the cryoprotective effect of low-density lipoprotein in Hariana bull semen. Four adult Hariana bull (3-4 years) after thorough andrological examination were selected for experiment. Semen was collected from each bull twice a week; total of 32 ejaculates (8 from each bull) were collected during the experiment. Collected sample from each bull was divided into 5 equal aliquots. Four aliquots were diluted separately with a Tris-based extender containing different concentration of low-density lipoproteins viz. 7% (T-1), 8% (T-2), 9% (T-3) and 10% (T- 4) as treatment while routinely used extender containing 20% hen egg yolk was used as control (C) to achieve final concentration of 125 million spermatozoa/ml. The diluted samples were cryopreserved and evaluated after 7 days. A significantly higher value of percent live sperms, sperm positive for hypo osmotic swelling test, sperms with intact acrosome and sperms exhibiting pattern F (uncapacitated sperm) were significantly higher in T-3 as compared to other treatment groups and control. The kinematic characters exhibited by sperm i.e forward progression %, VCL (μm/sec), VSL (μm/sec), VAP (μm/sec), Lin (%), Str (%), BCF (Hz) and ALH (μm) were significantly higher in T-3 as compared to other treatment group and control. In conclusion, extender containing 9% low density lipoproteins has better capacity to maintain the sperm character in post thaw Hariana bull semen and can be a preferred concentration to be utilized for cryopreservation with improved conception rate.

Effect of foot and mouth disease vaccination on seminal traits in pure Holstein Friesian bulls

The present investigation was carried out to study the effect of foot and mouth disease vaccination on seminal traits of 30 Holstein Friesian (HF) bulls, 3-5 years old, maintained at BAIF Central Research Station, Uruli Kanchan, Pune, India. The study period was from August to September 2013 and semen quality traits were evaluated on each bull maintained under identical feeding and management regimes. A total of 180 semen ejaculates were collected with one ejaculate from each bull at 3 days interval in a week at 15 days before, 15 and 30 days after FMD vaccination. Raksha Ovac vaccine of Indian Immunologicals Ltd. was used for vaccination purpose. Seminal traits like fresh and post thaw sperm motility, sperm concentration, semen volume, live and dead count, plasma membrane integrity were evaluated for all the ejaculates. Data on seminal traits were analysed by using one way ANOVA. Results revealed that vaccination had significant (P less than 0.05) effect on post thaw motility and highly significant (P less than 0.01) deleterious effect on HOS test, head and mid piece abnormalities; however no effect was noted on ejaculate volume, sperm concentration, initial motility, viability and tail abnormalities. The observations showed that vaccination alters the seminal traits of pure HF bulls.

Impact of holding and equilibration time on post-thaw quality of shipped boar semen

Cryopreservation of boar semen is of growing interest for breeding companies. Overnight-shipping of pre-diluted ejaculates to specialized laboratories offers a practicable method, but requires fine-tuned protocols. In this study, the impact of holding post shipping at 17°C for 2 or 24h (n=10 samples) and of equilibration in lactose-egg yolk extender without glycerol at 5°C for 2, 4, 24 or 48h (n=11 samples) before freezing was investigated. Sperm-rich fractions of ejaculates from 21 mature Pietrain boars were collected at a single boar stud. After pre-dilution (1+1, v:v) with Beltsville thawing solution, samples were sent to the laboratory. Temperature profiles during transport and initial equilibration time were recorded. Semen quality post-thaw (PT) was evaluated using CASA and flow cytometry. Holding of 2h after shipping resulted in higher sperm motility (P=0.013) and beat cross frequency (BCF; P=0.047) compared to 24h. Differences between both groups vanished with prolonged incubation at 38°C PT. Equilibration at 5°C for 4h yielded the highest motility and BCF, whereas the equilibration for 48h impaired sperm motility. Membrane integrity, mitochondrial activity and DNA fragmentation index were not affected by any protocol modification. In conclusion, processing of pre-diluted boar semen shipped overnight within 2h after arrival at the laboratory is preferred to 24h of additional holding at 17°C. Extending the equilibration period in lactose-egg yolk extender without glycerol at 5°C from 2h to 4h before freezing is recommended.

Boar variability in sperm cryo-tolerance after cooling of semen in different long-term extenders at various temperatures

This study investigated individual boar variability in the quality of pre-freeze (PF) and post-thaw (PT) semen cooled in different long-term (LT) extenders and for different holding times (HT). Sperm rich fractions were diluted with Androhep® Plus (AHP), Androstar® Plus (ASP), Safecell® Plus (SCP) and TRIXcell® Plus (TCP) extenders, stored for 2h at 17°C (HT 1) and additionally for 24h at 10°C (HT 2) and the samples were subsequently evaluated and frozen. Besides the analysis of CASA sperm variables, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), normal apical ridge (NAR) acrosome integrity, and viability (YO-PRO-1−/PI−) of sperm were assessed in the PF and PT semen. Results indicated that boar, extender and HT group affected the sperm quality characteristics. There were great variations in PMOT and the sperm motion patterns of the PF semen among the boars. Differences in the HT groups of the PF semen, with respect to the sperm membrane integrity, were less marked among the boars. Consistent variations in TMOT and PMOT in the PT semen were observed among the boars, being greater in the HT 2 group. Most of the CASA-analyzed sperm motion patterns were greater in the HT 2 group of the PT semen. Furthermore, sperm MMP, PMI and viability were greater in the HT 2 group of the PT semen in most of the boars, while consistent differences were observed among the boars for sperm NAR acrosome integrity in either HT group. The significant effect of the cryopreservation process on the sperm membrane proteome was evident from the number of protein bands, detected in the electrophoretic profiles of sperm of the HT 1 and HT 2 groups. The electrophoretic profiles of the PF and PT semen among boars with poor and good semen freezability, however, differed with respect to the abundance and types of sperm membrane-associated proteins. The overall results of this study provided evidence that there are differences among boars in response to the different cooling regimens, and that cooling of extended semen for a 24-h period at 10°C modulated the functions of sperm in an extender-dependent manner, rendering the cells less susceptible to cryo-induced damage. It is suggested that the findings of this study have the potential to improve the technology of boar semen cryopreservation.