Post-thaw Semen Characteristics Research Articles

Seminal characteristics and efficacy of egg yolk free semen extender on cryopreservation of Murrah buffalo bull semen

The experiment was undertaken to study the efficacy of egg yolk free semen extender on cryopreservation of buffalo semen. A total of 24 semen ejaculates from Murrah bulls (5 to 6 years) maintained at Central Semen Station, Anjora, Durg, Chhattisgarh were divided into two aliquots and were diluted using Tris-fructose-egg yolk-glycerol (TFYG) and egg yolk free commercial extender (EYFCE, AndroMed). The overall average Murrah bull fresh semen volume (mL), sperm concentration (million/mL), initial progressive motility (%), live sperm (%), abnormal spermatozoa (%), intact acrosome (%) and HOS reactive sperm (%) were 4.90±0.49, 1286.92±100.23, 75.00±0.67, 81.45±0.61, 10.70±0.51, 80.16±0.43 and 68.83±0.40, respectively. There was significant difference between bulls in semen volume (P<0.01), individual motility (P<0.01) and total sperm abnormalities (P<0.05). The semen extended with TFYG and EYFC extenders were cryopreserved in french mini-straw (0.25 mL) using programmable bio-freezer. The mean values of post thaw motility (%), live sperm (%), abnormal sperm (%), intact acrosome (%), HOST (%) of Murrah bull semen extended with TFYG and EYFCE were 45.83±0.77 vs 49.16±1.33, 57.41±0.54 vs 58.62±0.73, 12.54±0.46 vs 12.45±0.63, 69.62±0.82 vs 71.45±0.64 and 56.45±0.52 vs 57.37±0.55, respectively. Post thaw motility, live spermatozoa and intact acrosome were significantly higher (P<0.01) in frozen thawed semen extended with as EYFCE compared to TFYG in Murrah bull semen. It is concluded that EYFCE results in improvement in post thaw semen characteristics viz. post thaw motility, live sperm percent and intact acrosome.

Effect of Membrane Stabilizers on Semen Quality and Sperm Membrane Protein Expression During Cryopreservation of Goat Semen

BACKGROUND: Semen cryopreservation is a complex process during which there is alteration in the expression of sperm and seminal plasma proteins, molecular weight of protein or loss of membrane proteins during the process. In order to compensate for these changes, different membrane stabilizers are used in freezing semen extenders. However, there is scarcity of such studies during cryopreservation of goat semen. OBJECTIVE: To investigate the effect of membrane stabilizers on sperm membrane protein expression during cryopreservation of goat semen. MATERIALS AND METHODS: A total of 36 semen ejaculates from nine Assam Hill Goat bucks aged 2 to 2.5 years was collected by artificial vagina method. Three membrane stabilizers, each at two different concentrations viz. 50 and 80 mM sucrose, 50 and 100 mM trehalose, and 100 and 150 ng/mL IGF-1 (insulin-like growth factor 1 protein) were added to Tris-citric acid fructose egg yolk glycerol (TCFEYG) extender and semen samples were cryopreserved. The sperm membrane protein profile was studied in fresh and cryopreserved semen by SDS-PAGE. RESULTS: SDS- PAGE of sperm membrane extract of fresh semen revealed the presence of 24 protein bands with molecular weights ranging from 10 kDa to 240 kDa. Samples supplemented with 50 mM sucrose and 80 mM sucrose revealed 21 protein bands with molecular weights ranging from 10 kDa to 240 kDa. All the 21 protein bands were same as those observed in the sperm membrane of fresh spermatozoa, except that the 23 kDa, 29 kDa and 42 kDa bands were absent in frozen semen. Similarly, frozen semen extended with 50 mM trehalose and 100 mM trehalose revealed 22 protein bands with molecular weights ranging from 10 kDa to 240 kDa, but lacking the 29 kDa and 42 kDa bands. Proteins with molecular weights of 29 kDa, 130 kDa and 240 kDa were absent in frozen semen supplemented with 100 ng/mL IGF-1 and 150 ng/mL IGF-1. CONCLUSION: The present study revealed that supplementation of tris basic extender with trehalose at 100 mM and or IGF-1 at 100 ng/mL or 150 ng/mL improves the post-thaw semen characteristics and protects certain fertility related sperm membrane proteins.

Effects of age, size and season on cryotolerance of dog semen – A retrospective analysis

In a retrospective analysis of 508 ejaculates from 297 dogs, efficiency of semen cryopreservation and effects of age, season and dog size on characteristics of fresh and cryopreserved semen were evaluated. Volume of the sperm-rich ejaculate fraction increased to 10 years of age, decreased in 10 and 11-year old dogs (P < 0.001) but did not differ among seasons. Total sperm count was less in 10 and 11-year-old dogs (P < 0.001). The percentage of progressively motile, membrane-intact and morphologically normal spermatozoa before cryopreservation was least in 10 and 11-year-old dogs (P < 0.001). Cryopreservation resulted in less progressively motile spermatozoa (P < 0.001) with this being most pronounced in 10 and 11-year-old dogs (cryopreservation x age P = 0.004). The cryopreservation-induced decrease in morphologically normal spermatozoa (P < 0.001) was not affected by dog body weight. Number of cryopreserved AI doses differed among age groups (P < 0.001) and was less in 10 and 11-year-old dogs (median 1.5) compared with younger dogs (6–7 years, median 4.3). When ejaculates were grouped by a threshold of ≥ 35% progressively motile spermatozoa after freezing-thawing, 86.5% of all ejaculates were greater the threshold but this percentage decreased to 66% in 10 and 11-year-old dogs (P < 0.001). In conclusion, sperm cryotolerance is consistent for much of a dog´s lifespan but decreases after a certain age. Dog semen can be cryopreserved successfully throughout the year. Post-thaw semen characteristics were not different among cryopreserved ejaculates obtained from dogs differing in body weight.

Seasonal Influence on Post Thaw Seminal Attributes and Conception Rate from Semen of Jaffarabadi Buffalo Bulls

Background: The quality of the frozen thawed semen is one of the most powerful factor affecting the conception rate in a buffaloes. The season may have its effect on post thaw seminal attributes and fertility. Therefore, current study was aimed to investigate the effect of winter and summer seasons on post thaw seminal attributes and conception rate from semen of Jaffarabadi buffalo bulls.Methods: The study was conducted on four Jaffarabadi breeding bulls, 5-6 years old, to know the post-thaw semen characteristics based on total of 192 semen ejaculates evaluated and cryopreserved over one year period. Result: The mean percentages of post-thaw motility, live sperm, abnormal sperm, HOST reactive sperm, acrosomal integrity and first AI conception rate for semen cryopreserved during winter were 58.85±0.56, 67.06±0.77, 8.62±0.43, 58.52±0.67, 75.22±0.27 and 45.75±0.21, respectively, whereas, corresponding values for semen cryopreserved during summer were 56.35±0.43, 65.62±0.76, 9.08±0.51, 55.43±0.60, 74.29±0.22 and 43.50±0.11. The semen quality of post thawed samples were observed better during winter compared to summer season for artificial insemination with satisfactory first AI conception rate.

Investigation of the changes observed in scrotal circumference, and native and post-thaw semen characteristics in karayaka rams during the breeding and nonbreeding seasons

The aim of the present study was to investigate and compare the impact of the breeding (BS) and non-breeding (NBS) seasons on scrotal circumference, and native and post-thaw semen characteristics in Karayaka rams. Six (6) Karayaka rams were used as semen donors, and ejaculates were collected with the aid of an artificial vagina during the BS (October-November) and NBS (April-May). Interestingly, sperm freezability was found to be higher in the NBS, when compared to the BS, and the sperm motility rate (36.00±2.74%, 55.0±3.24%) and percentage of abnormal sperm (44.00±1.90%, 57.00±2.09%) were found to significantly differ with season (P&lt;0.05). In conclusion, the post-freezing sperm motility was better during the non-breeding season. Post-thaw sperm abnormality was higher during non-breading season. However, if ram semen is used fresh for AI, it can be collected and used during both during the breeding and non-breeding season.

Variance component estimates for post-thaw sperm variables measured by computer assisted semen analyzer for inbred and non-inbred Hereford bulls

Inbreeding has been shown to have negative effects on animal reproductive performance. It, however, has not been documented as to the extent that inbreeding impacts sperm cell characteristics especially after cells have undergone cryopreservation. In the present study, therefore, there was evaluation of sperm motion and size characteristics from two inbred lines and non-inbred Hereford bulls using Computer Assisted Sperm Analysis (CASA). Pre-frozen semen samples were obtained from Line 1, Prospector, and a non-inbred Hereford bulls. Average inbreeding of each group was 27%, 45%, and 0%, for Line 1, Prospector, and non-inbred groups, respectively. For each bull, two straws (0.5 ml) were evaluated, within each straw two slides with ten fields per slide analyzed with the CASA. Cells were analyzed by mixed model analysis using a model that contained line as fixed effect and individual bull, straw, slide, and fields are random effects. No significant differences between lines of bulls were found for motility or progressive motility. For the head elongation (ELON), and head area (AREA) significant differences existed between lines. Mean separation indicated that Line 1 group had a larger head area and the heads were not as elongated as the Prospector and non-inbred group cells. The lack of significant differences for motility and progressive motility across lines was surprising and indicates there was no inbreeding depression for post-thaw semen characteristics. One explanation for this could be that indirect selection pressure was placed on these characteristics as inbreeding was increased.

Effect of sericin supplementation on heat shock protein 70 (HSP70) expression, redox status and post thaw semen quality in goat

Cryopreservation results in substantial deterioration of heat shock protein 70 (HSP70) and ultra-structural changes in sperm organelles, resulting in a marked reduction in post-thaw semen quality. The present study was aimed to explicate the effect of sericin supplementation on expression profile of HSP70, redox status and post-thaw semen quality in Barbari goat. Five Barbari bucks were used to collect thirty semen ejaculates by using artificial vagina and each ejaculate was divided into three aliquots to which sericin was supplemented at 0% (Control), 0.25% (T1) and 0.50% (T2). Further, extended semen samples were equilibrated followed by their cryopreservation. Post-thaw semen characteristics, redox status of seminal plasma, enzyme leakage and HSP70 gene/protein expression in spermatozoa were assessed in all the groups. Per cent progressive motile spermatozoa, spermatozoa having intact plasma membrane (HOST + ve) and intact acrosomes in post-thaw spermatozoa were significantly (p < 0.01) higher in T1 and T2 as compared to control. A significant (p < 0.01) reduction in abnormal spermatozoa was found in T1 as compared to T2. Sericin supplementation significantly (p < 0.05) improved the antioxidative status (SOD, GST, CAT), reduced lipid peroxidation (MDA) and also prevented enzyme (ALT, LDH) leakage as compared to control samples. qRT-PCR results revealed that HSP70 mRNA expression was significantly (p < 0.01) upregulated in T1 and T2 group as compared to control. The positive effect of sericin on expression of HSP70 was further confirmed by immunoblotting followed by densitometry revealing higher expression in T1 and T2 compared to control. Inclusion of 0.25% w/v sericin in semen extender ameliorated the post-thaw semen quality by improving antioxidative status and minimizing the leakage of intracellular enzymes. Sericin supplementation had a beneficial effect on HSP70/HSP70 mRNA expression either by induction or by protection of HSP70/HSP70 mRNA as evident from the gene expression and immunoblotting studies.

Characteristics and freezability of Assam Hill goat semen

Assam Hill goat (AHG) is an important goat germplasm found in Assam and its adjoining areas of India. The study was designed with an objective to study the semen characteristics and freezability of AHG buck semen using Tris -Egg yolk-Citrate-Fructose diluent. The mean values of fresh semen characteristics in AHG bucks viz., ejaculate volume (ml), initial sperm motility (%), sperm concentration (x106/ml), live sperm (%), sperm abnormality (%), HOST-reacted sperm (%) and intact acrosome (%) recorded were 0.39 ± 0.01, 77.97 ± 0.73, 3201.00 ± 143.78, 83.02 ± 0.65, 7.66 ± 0.73, 66.95 ± 0.74 and 93.34 ± 0.51, respectively. Mean values for post-thaw semen characteristics i.e., sperm motility (%), live sperm (%), HOST-reacted sperm (%) and intact acrosome (%) were 55.39 ± 0.97, 71.01 ± 0.78, 54.77 ± 0.55 and 82.16 ± 0.43, respectively. It can be concluded that AHG bucks donate acceptable quality of semen which can be frozen successfully in Tris-Egg yolk-Citrate-Fructose diluents for using in Artificial Insemination.

Effects of single layer centrifugation (SLC) on bull spermatozoa prior to freezing on post-thaw semen characteristics.

Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen-thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species-specific colloid, Bovicoll. After centrifugation for 20min at 300g, the resulting sperm pellet was resuspended in OPTIXcell® (IMV Technologies, l'Aigle, France); the SLC-selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC-treated samples had a higher percentage of live, superoxide-positive spermatozoa than uncentrifuged samples (27.9±5.1% versus 21.7±6.7%; p=.03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9±8.2% versus 40.5±15.1%; p=.03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non-treated samples (42.0±8.5% versus 55.9±14.4%; p=.04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide-positive spermatozoa and live, hydrogen peroxide-negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC-selected spermatozoa compared to unselected samples.

Seminal plasma hormonal profile of Arabian stallions that are classified ‘good’ or ‘poor’ for semen freezing

ObjectiveTo relate seminal plasma reproductive and metabolic hormone profile to both semen freezability and post-thaw semen characteristics. MethodsSemen was collected from each Arabian stallion (N = 16) for five consecutive weeks and evaluated before conducting this study. Seminal plasma was collected during semen processing for measuring leptin, insulin, Insulin like growth factor-I (IGF-1), cortisol, testosterone, estradiol and thyroid hormones. Stallions were classified into good (N = 10) and poor freezer (N = 6). Semen post-thaw motility (%), viability index, membrane and acrosome integrity (%) were also evaluated. ResultsGood freezer stallions had significantly (P = 0.0001) high cortisol, estradiol, insulin, IGF-1, T3 and T4, but significantly low leptin (P = 0.003). Post-thaw sperm viability index (P = 0.0001), membrane integrity (%) (P = 0.002) and motility (%) (P = 0.0001) at 0, 1, 2 and 3 h of good freezer stallions was significantly high compared to poor freezers. Testosterone, insulin and IGF-1 in seminal plasma of good freezer stallions had significant (P < 0.01) positive correlation with sperm post-thaw viability index but estradiol and insulin in seminal plasma of poor freezers correlated with sperm post-thaw viability index. Within good freezer stallions, testosterone in seminal plasma correlated positively with both viability and acrosome integrity but within poor freezer stallions it had a negative correlation with both of them. Individual variation existed between animals and significantly affected seminal plasma hormones and semen post-thaw characteristics. ConclusionGood freezer stallions could be selected depending on assaying seminal plasma hormones. Both estradiol and IGF-1 could be used as semen extender supplementation to improve sperm post-thaw motility, viability and acrosome integrity.

Role of amino acids as additives on sperm motility, plasma membrane integrity and lipid peroxidation levels at pre-freeze and post-thawed ram semen

The possibility of including amino acids for cryopreservation of ram semen to improve the quality of frozen semen was explored in this study in sheep model. 24 samples were collected in triplicate from 8 rams of 2–3 year old Bannur cross bred rams maintained at the Institute Experimental Livestock Unit. Semen was diluted in tris-egg yolk glycerol diluent and made into 7 aliquots as follows: aliquot 1 served as control, “l-alanine” was added at 100 and 135mM in the aliquots 2 and 3, “l-glutamine” was added at 20 and 25mM in the aliquots 4 and 5 and “l-proline” was added at 25 and 50mM in the aliquots 6 and 7, respectively. Diluted semen was filled in 0.25ml French straws and frozen in LN2. Inclusion of “l-proline” and “l-glutamine” in the diluent increased the percent live sperm (P<0.001), total motility (P<0.05) and maintained higher functional membrane and acrosomal integrity (P<0.001) by decreasing lipid peroxidation (P<0.001) compared to the control group. In contrast, “l-alanine” decreased the percentage of total motility, fast progressive spermatozoa and increased (P<0.01) the percentage of immotile spermatozoa. It can be concluded that 20mM “l-glutamine” and 25mM “l-proline” can be used as semen additive to freeze ram semen as they prevented cryoinjuries to sperm and improved the pre-freeze and post-thaw semen characteristics.

Effect of amino acids on sperm motility, velocity parameters, plasma membrane integrity and lipid peroxidation levels at cooled and post–thawed ram epididymal semen

This study is focused to look into the possibility of including amino acids during cryopreservation to improve the quality of frozen semen in sheep. Cauda epididymal semen was collected from 2-3 year-old Bannur crossbred rams slaughtered at the local abattoir. The semen samples were diluted in tris egg yolk glycerol diluent along with different additives and made into 7 aliquots. Aliquot 1 served as control, L-alanine was added @ 100 and 135mM in the aliquot 2 and 3, L-glutamine was added @ 20 and 25mM in the aliquot 4 and 5 and L-proline was added @25 and 50mM in the aliquot 6 and 7, respectively. Diluted semen was filled in 0.25 ml French straws, sealed manually and equilibrated at 4oC for 2 h, cooled in LN2 vapour before being plunged into LN2 till further evaluation. Inclusion of L-proline and L-glutamine in the diluent significantly increased the percent live sperm, total motility, lipid peroxidation and maintained higher functional membrane and acrosomal integrity than the control group. In contrast, L-alanine decreased the percentage of total motility, fast progressive spermatozoa and increased the percentage of immotile spermatozoa. It can be concluded that 20mM L-glutamine and 25mM L-proline can be used as semen additive to freeze ram epididymal semen as they prevented cryo injuries to sperm and improved the pre-freeze and post-thaw semen characteristics.

Environmental and male variation factors of freezability in rabbit semen

The aim of this study was to analyze the environmental and male effects that could have an influence on sperm freezability using a recursive model. A total of 853 ejaculates from 217 males belonged to a paternal rabbit line were collected and frozen. Six different traits were evaluated: the sperm concentration (106 spermatozoa per mL), the acrosome integrity on fresh (%) and frozen-thawed semen (%), the sperm motility on fresh (%) and frozen-thawed semen (%), and the percentage of viable sperm on frozen-thawed semen (%). In addition, two synthetic traits were computed, the relative reduction of acrosome integrity (%) and relative reduction of motility (%) after the freezing-thawing process. A multiple-trait recursive model was used to analyze the relationships between the semen traits considered. For the fixed effects studied, the season had the highest effect on postthaw semen characteristics. Results of the analysis of recursive coefficients showed that fresh semen concentration and motility influence the future freezability of the semen. All traits studied presented moderate repeatabilities ranging from 0.11 to 0.38. These results provide conclusive evidence that sperm freezability in rabbits could be heritable. Regarding male correlations, there were large positive male correlations between fresh traits (rm = 0.77 to 0.57), and between direct frozen-thawed traits (rm = 0.72 to 1). Male effects on fresh and direct frozen-thawed traits were generally positively correlated. This correlation was moderate to high for fresh semen motility with all frozen-thawed traits (rm = 0.41 to 0.74) and for frozen-thawed semen and all fresh traits (rm = 0.5 to 0.74), these results suggest that these traits could be genetically related. Further studies involving more males and ejaculates should be conducted in the future to estimate the heritabilities and genetic correlations of postthaw semen traits in rabbits.

Characteristics of cryopreserved semen from men with lymphoma.

This study compared the pretreatment semen quality in patients with Hodgkin's disease and non-Hodgkin's lymphoma with a group of healthy donors. We also examined the differences in prefreeze and postthaw semen quality among the different stages of Hodgkin's disease. The study included 89 patients with Hodgkin's disease, 18 with non-Hodgkin's lymphoma, and 50 healthy sperm donors. In patients with Hodgkin's disease, the prefreeze and postthaw semen characteristics were significantly lower than those of the healthy donors. Similar results also were seen in patients with non-Hodgkin's lymphoma. No significant differences in the prefreeze semen quality were seen in patients with different stages of cancer. Patients with Hodgkin's disease and non-Hodgkin's lymphoma in our study had poor semen quality when compared with healthy donors both before and after cryopreservation. As cancer therapy significantly impairs reproductive potential, sperm banking should be offered to these men before the start of their therapy.

Why Cancer Patients Request Disposal of Cryopreserved Semen Specimens Posttherapy: A Retrospective Study

Objective: To determine why patients with cancer stop storing semen in a sperm bank program. Design: Retrospective study. Setting: Hospital andrology laboratory. Patient(s): Cancer patients (n = 56) who discontinued sperm storage. Intervention(s): A database of 342 patients with cryopreserved sperm was searched for disease diagnosis, marital status before and after diagnosis, type of therapy, number of specimens banked, interval between diagnosis and sperm banking, and postthaw semen characteristics. Patients discontinuing storage were surveyed. Main Outcome Measurement(s): Reasons for discontinuing storage and clinical correlation of the decision. Result(s): Reasons included patient death (n = 21); fertility but no plans for more children (n = 23); good sperm quality (n = 8); and no plans to have children (n = 4). Patients were similar in age, number of specimens, and interval between diagnosis and treatment, but they showed significant differences in type of treatment and time in the program. Cost of cryopreservation and specimen storage was not cited. Conclusion(s): Most patients decided to discontinue sperm banking because either they regained fertility or had improved semen quality. Sperm banking should be strongly recommended for all patients with malignant diseases who may wish to have children, even if they eventually decide that the specimens are not needed.

Evaluation of Spermatological Parameters Used to Predict the Fertility of Frozen Bull Semen

Post-thaw motility, velocity and acrosome integrity of frozen semen were determined in 18 bulls with varying fertility (average non-return rates: 71.3 (+/- 2.8)--range: 65.2-75.7). Five semen straws were investigated from each bull. The average values for sperm motility (percentage motile spermatozoa), sperm velocity (graded from 0-3) and acrosome integrity (proportion of spermatozoa with intact acrosome) were 67.5%, 2.5 and 79.3%, respectively. Significant correlations were found between sperm motility and velocity, but not between sperm motility and acrosome integrity. Both sperm motility and velocity were significantly related to bull fertility. It was concluded that of the post-thaw semen characteristics investigated in this study these 2 parameters provided a reliable basis for prediction of bull fertility.

Prefreezing and post-thaw semen characteristics of five ram breeds collected by electroejaculation

Rams representing five breeds were electroejaculated twice weekly, during a three-week collection period. Ejaculates were evaluated for volume and concentration before freezing and for rate of motility and percentages of motile and abnormal cells both before and after freezing. Interactions between breed and collection period were evident (P<0.05) for semen volume and post-thaw values for rate of motility and percentage motile cells. Breeds differed (P<0.05) in these traits during some periods. In contrast, pre-freezing observations of rate of motility, percentage motile and abnormal cells and post-thaw percentage abnormal cells did not differ (P>0.15) among breeds. Sperm concentration per ejaculate tended to vary (P=0.11) among breeds. Semen characteristics frequently varied across collection periods. Rams within a breed differed (P<0.01) in all semen traits except post-thaw rate of motility and percentage motile cells. Semen was negatively affected by the freezing and thawing procedure. Ram within a breed and ejaculate within ram should be considered when selecting electroejaculated semen for freezing and subsequent use in artificial insemination.