Cryopreservation Of Bull Semen Research Articles

Application of High Hydrostatic Pressure (HHP) to Improve Cryopreservation of Young Bull Semen

Abstract The objective of the study was to determine the effect of high hydrostatic pressure (HHP) on quality of cryopreserved semen of young bulls. Semen for this study was collected from 8 bulls aged between 13 and 18 months at monthly intervals, from June to September. After collection, semen was diluted in a commercial Bioxcell® extender (one part at 1:1 and a second part to give a sperm concentration of 20 million/0.2 mL), filled into straws and treated with HHP at 30 MPa for 90 min. After HHP treatment, pre-diluted semen (1:1) was diluted to a sperm concentration 20 million/0.2 mL and filled into straws. In addition, part of the semen diluted to a concentration of 20 million/0.2 mL was not treated with HHP (control). All of it was held at +4°C and frozen in a freezer after 2.5-h equilibration. Semen was thawed in a water bath at 38°C and subjected to estimation of the percentage of motile sperm both subjectively and using a computer-assisted semen analyzer and cytometric assessment of sperm cell membrane integrity. Subjective motility and fast progressive motility were significantly higher with pre-diluted (1:1) and HHP treated semen compared to control (P<0.05). No significant differences were observed in percentage of membraneintact spermatozoa between control and experimental groups. Additionally, the influence of HHP on the sperm of individual bulls was assessed. In bull number 2, the HHP treatment after semen pre-dilution significantly improved progressive motility from 54.1 to 63.4 percent (P<0.05). In bull number 4, the HHP treatment after semen pre-dilution significantly improved subjective motility, rapid motility and progressive motility by 12.5, 16.8 and 16.3 percent, respectively (P<0.05). No effect was seen for 6 bulls. It is concluded that for some bulls, the application of HHP before semen freezing may improve the cryopreservation outcome. However, this requires further research in this area, also to determine the fertilizing capacity of bull semen exposed to high hydrostatic pressure.

Commercial application of flow cytometry for evaluating bull sperm

Artificial insemination using semen from genetically superior sires remains one of the most effective biotechnologies ever commercialized for animal breeding purposes. Genetic progress, however, cannot begin until conception occurs. Processing laboratories that provide cryopreserved bull semen for commercial use depend on in vitro assays of semen quality to identify samples that are expected to result in less than desirable conception rates. These identified samples are discarded, rather than released to salable inventories, with the desired effect of minimizing variance in field fertility among both sires and individual collections. Although the industry was successfully founded on subjective assessment of motility and acrosome integrity, flow cytometric and computer-assisted sperm analysis offer more objective and repeatable measures of sperm quality attributes. Albeit more expensive to implement, the increased precision and repeatability when using these objective assays lends to greater confidence in the accuracy of decisions for individual collections and (or) bulls. The efficacy of a quality control program is evidenced by the range in sire fertility estimates calculated from field fertility data, which have historically indicated >90% of all sires achieve fertility deviation within ±3% points of the breed average. This impressive precedent implies somewhat limited opportunity for transition to objective assessments to have a meaningful impact on an already narrow range of fertility distributions. Nonetheless, flow cytometric assessments of novel attributes of sperm quality hold promise for detection of truly sub-fertile sires (deviations < −3) that presently elude detection with use of existing semen bioassays.

The Influence of Zinc and Heavy Metals in Feed and Water on the Quality of Cryopreserved Bull Semen

Abstract This study aimed to assess the influence of different concentrations of zinc (Zn), lead (Pb), mercury (Hg) and cadmium (Cd) found in the feed and water provided to bulls on the concentrations of these metals in cryopreserved bull semen, and to determine their influence onto semen quality parameters. Correlations between heavy metal concentrations in the semen and the quality parameters of semen as estimated by computer-assisted sperm analysis (CASA) and flow cytometry (FC) methods were determined. A total of 40 cryopreserved semen samples originating from bulls housed in 4 different centers for artificial insemination (A, B, C and D) were examined, making a total of 160 samples. The concentrations of metals and semen quality parameters were determined in cryopreserved semen of 10 bulls from each center, namely 4 samples from each bull. Concentrations of Zn, Pb, Hg and Cd in hay and concentrated feed were within the allowed limits as proposed by the National Research Council (NRC, 2000). A strong negative correlation was detected between curvilinear velocity (VCL) and Zn concentrations in the semen (P &lt; 0.01; r = -0.772) in group D, and a positive correlation of VCL with Pb concentrations (P &lt; 0.05 and r = 0.718) in group B. Mercury concentrations in cryopreserved semen correlated negatively to the percent of live sperm cells with intact acrosomes (V/IA: P &lt; 0.05; r = -0.640) and positively with the percent of dead sperm cells with damaged acrosomes (D/DA: P &lt; 0.01; r = 0.766) in group D. This finding confirms the hypothesis that Hg, even at low concentrations, may cause acrosome damage.

Cholesterol-cyclodextrin complex as a replacement for egg yolk in bull semen extender: sperm characteristics post-thawing and in vivo fertility

Egg yolk, a major semen extender constituent, lacks a defined composition, therefore, there are biosecurity concerns with use of egg yolk. Cryopreservation of bull semen without inclusion of animal protein in the semen extender, therefore, is an important consideration. Cholesterol may be delivered and incorporated into the sperm plasma membrane by cyclodextrins to protect sperm during cryopreservation. The aim of this study was to determine suitability of a cholesterol-cyclodextrin semen extender, without inclusion of egg yolk, for cryopreservation of bull semen. Bull semen was collected and cryopreserved in either egg yolk or with inclusions of three different concentrations of cholesterol-cyclodextrin complex (0.5, 1 or 2 mg/mL semen) in Tris-glycerol (TG) extender. Sperm motion characteristics examined using the computer-assisted sperm analysis, and plasma membrane and acrosome integrity examined using flow cytometry, were similar for all extenders. The inclusion of the greatest concentration of cholesterol-cyclodextrin complex (2 mg/mL semen) followed by dilution in TG extender resulted in lesser pregnancy rates (P < 0.05). There was a pregnancy rate of as great as 56 % when sperm cryopreserved in 0.5 mg/mL cholesterol-cyclodextrin Tris-glycerol extender were used for artificial insemination following imposing of a hormonal treatment regimen for synchrony of timing of ovarian functions among cows for conducting fixed-time artificial insemination (FTAI). Results indicate cholesterol-cyclodextrin Tris-glycerol extender, with a chemically defined composition and without inclusion of egg yolk, may be used to cryopreserve bull sperm with there being acceptable pregnancy rates when this semen is used for FTAI.

Heat treatment of milk potentiates its antioxidant activity through sulfhydral groups formation and improves buffalo bull semen cryopreservation in a milk-based extender

Heat treatment of milk potentiates its antioxidant activity through sulfhydral groups formation and improves buffalo bull semen cryopreservation in a milk-based extender

Cryopreservation of Indigenous bull semen (Zobawng) of Mizoram: Effect of fructose on extender

A total of 36 ejaculates were collected from three indigenous bulls (Zobawng) of Mizoram. The semen samples were evaluated to find out the effects of fructose i.e. Tris egg yolk citric acid fructose glycerol (TEYCAFG) and Egg yolk sodium citrate glycerol (EYSCG) extenders on freezability of indigenous bulls’ (Zobawng) semen of Mizoram in terms of sperm motility, per cent live sperm, HOSST reacted spermatozoa and acrosomal integrity. The mean sperm motility, per cent live sperm, HOSST and acrosomal integrity in TEYCAFG extender at pre and post freezing were (69.53±0.31 and 45.36±0.59%), (76.36±0.35 and 53.03±0.45%), (67.91±0.45 and 44.92±0.56%) and (75.17±0.58 and 63.25±0.56%) respectively, and in EYSCG extender were (45.67±0.48 and 33.08±0.36%), (54.56±0.41 and 38.08±0.28%), (47.86±0.44 and 34.61±0.40%) and (57.22±0.31 and 45.89±0.48%) respectively. The mean motility, live sperm, HOSST and acrosomal integrity in both TEYCAFG and EYSCG extenders were significantly (P

Sodium caseinate improves longevity and fertility of frozen bull semen

This study aimed to evaluate the effect of sodium caseinate added into freezing extender on the sperm parameters of cryopreserved bull semen and in vitro and in vivo fertility. One ejaculate of 30 bulls was used and processed using Botu-Bov (Botupharma, Botucatu, Brazil) with the addition of 20% egg yolk (EY) or 15% egg yolk with 2% sodium caseinate (EY + SC), subsequently submitted to freezing. Semen from both groups were evaluated immediately after thawing (T0) and after thermic stress at 37 °C for 90 min (T90), for sperm kinetics, by CASA method, and plasma membrane integrity (PMI), superoxide (O2−) concentration and high mitochondrial potential (HMP) by flow cytometry. In vitro fertilization (IVF) was performed to assess embryo cleavage rate on day 3, and blastocyst rate on day 8. The in vivo fertility test was performed using fixed-time artificial insemination (FTAI). In sperm evaluation, trajectory velocity, linear velocity, curvilinear velocity, and lateral head movement were higher (P < 0.05) in EY + SC at T0. At T90, while rectilinearity and linearity did not differ between EY and EY + SC (P > 0.05), the other parameters evaluated were higher in EY + SC. Similarly, the integrity of the plasma and acrosomal membranes (iPAM) was higher (P < 0.05) at T90 in EY + SC, but did not differ (P > 0.05) between the groups at T0. For O2− and HMP, the values were lower (P < 0.05) in EY + SC group in both moments; furthermore, EY + SC showed higher cleavage and blastocyst rates in IVF. Likewise, pregnancy rates by FTAI were higher (P < 0.05) in the EY + SC group. In conclusion, the addition of sodium caseinate into freezing extender improves sperm parameters of frozen-thawed bull semen and fertility rates on during in vitro and in vivo tests.

Cryopreservation Induces Alterations of miRNA and mRNA Fragment Profiles of Bull Sperm.

Although cryopreservation of bull semen is widely used commercially in the livestock breeding industry, cryopreservation results in low fertility of bull sperm. As an important regulatory factor, the alteration of small non-coding RNA (sncRNA) profile during cryopreservation of bull sperm is not yet completely known. In the present study, we sequenced sncRNAs of frozen and fresh sperm to study the link of alteration of the sncRNA profiles (particularly in miRNAs and mRNA fragments) with low sperm fertility caused by cryopreservation. We identified 55 miRNAs and 526 mRNA fragments differentially expressed (DE) between frozen and fresh sperm. Subsequently, the functional analysis revealed that targeted genes of DE miRNAs in sperm had roles in the fertilization, ATP, and apoptosis. Instead, targeted genes of DE miRNAs in cow metaphase II oocyte were significantly enriched in the MAPK signaling pathway, autophagy-animal pathway, and mitophagy-animal pathway. Additionally, biological processes of DNA repair, spermatid development, response to temperature stimulus, and cellular response to DNA damage stimulus were enriched by mRNA fragments. In conclusion, we found that DE miRNAs or DE mRNA fragments in cryopreservation may influence the fertility of sperm, these findings will provide the reference to improve the cryopreservation technology of bull semen.

Effects of Leucosporidium-derived ice-binding protein (LeIBP) on bull semen cryopreservation.

We examined the effect of ice‐binding protein derived from Leucosporidium (LeIBP) on the cryopreservation of bull semen and compared it with that derived from previously reported Antifreeze Protein III (AFPIII). Six concentrations of LeIBP (10–1 ~ 104 μg/ml) and AFPIII (10–1 ~ 104 μg/ml) were added to the bull semen extender, respectively. Sperm kinematic parameters were measured to examine sperm toxicity and cryopreserved sperm quality. Measures of antioxidant activity such as superoxide dismutase (SOD), reduced glutathione/oxidative glutathione (GSH/GSSG), and total antioxidant capacity (TAC) were analysed to identify the effect of LeIBP on sperm quality. In addition, sperm viability was analysed using a flow cytometer and fluorescence microscope by SYBR14/PI staining. The results showed that the LeIBP groups (0.1, 1 and 10 μg/ml) were less toxic, and the quality of the sperm were dramatically improved in the extenders containing 0.1 μg/ml LeIBP among concentrations of LeIBP and AFPIII. The SOD activity of LeIBP was greater than that of AFPIII and control. In addition, sperm viability was enhanced in the LeIBP‐treated group. In summary, LeIBP is a useful cryoprotective adjuvant for bull sperm cryopreservation, and the most efficient concentration of LeIBP is 0.1 μg/ml.

In vitro assessment of egg yolk-, soya bean lecithin- and liposome-based extenders for cryopreservation of dairy bull semen

The study was conducted to compare the effect of four commercially available extenders (Triladyl®- egg yolk-based; Andromed® and Bioxcell®-plant based and Optixcell®-liposome-based) on post‐thaw sperm quality and functionality variables evaluated using computer‐assisted sperm analysis and flow cytometry. A total of 30 ejaculates from five bulls were analysed. With use of Triladyl®, sperm had a greater post-thaw total motility than with use of Bioxell® and Optixcell® but there was no difference as compared with use of Andromed® with the greatest (P < 0.05) percentage of progressively motile cells. With use of Optixcell®, there was a greater (P < 0.05) percentage of sperm with an intact membrane than with use of Triladyl® and Bioxcell®, but values were similar with use of Andromed®. Acrosome damage in semen preserved with use of Optixcell® was less than with use of Bioxcell® and Andromed®. With use of Optixcell®, there was a greater percentage of viable spermatozoa with a lesser lipid disruption (P < 0.05) when compared with the other extenders. Production of peroxides was greater for sperm cryopreserved with use of Triladyl® and Optixcell® while less superoxide was produced in the samples cryopreserved with the egg yolk-based extender. Optixcell® appears to be a promising alternative to replace traditional egg yolk extenders. With use of Optixcell®, however, there were greater peroxide concentrations after thawing. With use of Andromed®, there were similar results as with use of Optixcell®, therefore, it could be an effective substitute for egg-yolk based media due to the greater proportion of highly and progressively motile spermatozoa at thawing.

Bacteriospermia and Sperm Quality of Cryopreserved Bull Semen Used in Artificial Insemination of Cows in South Wollo Zone, Ethiopia.

The objectives of this trial were to estimate prevalence of bacteriospermia, to determine the bacterial load, and to isolate the types of bacteria as well as to assess the association between bacterial load and sperm quality traits in cryopreserved bull semen in field conditions in the South Wollo Zone. A total of 309 cryopreserved straws of semen from the Holstein Friesian (HF)-cross bull (n = 180 straws) and pure Jersey bull (n = 129 straws) were investigated. Bacteriological assessments of the presence of aerobic bacteria, estimation of bacterial count and bacterial isolation, as well as semen quality were performed. Aerobic bacterial contamination was prevalent in 38.8% of the semen straws. No significant difference in the prevalence of bacteriospermia was observed among bulls although the HF-cross bull had a higher prevalence (40.0%). But, significant difference in prevalence of bacteriospermia was found among semen ejaculates of the same bull. The risk of bacteriospermia in the HF-cross bull was higher (Odds ratio = 1.86, 95% CI = 0.168–20.26) compared to Jersey although not significant. Overall average bacterial load of 50.38 ± 16.29 colony-forming units (CFU)/ml (from nil to 1318.20 CFU/ml) was found. No significant difference in bacterial count among bulls and their ejaculates was observed. Moreover, correlation analysis revealed that the proportions of motility, live, and normal morphology were negatively influenced by an increase in the bacterial contamination of semen. In this study, three isolates of coagualse-negative Staphylococcus species and one isolate of Corynebacterium species were found. Average percentages of sperm motility (48.35 ± 1.23), live (66.08 ± 1.0), and normal morphology (80.62 ± 1.24) were observed. It was concluded that cryopreservation does not guarantee the quality of semen from bacterial contamination. Hence, meticulous care should be adopted to prevent contamination of semen by bacteria during collection, transportation, processing, and storage times.

The use of nanosized liposomes from vegetable phospholipids in combination with albumine and some polysaccharides as cryoprotective agents in the course of cryopreservation

It is known that nanosized liposomes from vegetable and animal phospholipids promote survival and functional adequacy of animal cells in the course of cryopreservation. It is also known that albumine and some polysaccharides have membrane-protective properties. In this paper we investigated whether there is a synergism of cryoprotective action between soybean liposomes and macromolecular compounds: albumine, sodium alginate, methylcellulose, carageenan and hyaluronic acid. To determine the cryoprotective effect we carried out cryopreservation of bull semen using a standard procedure in the presence of the substances under investigation. Albumine and sodium alginate demonstrated the most considerable cryoprotective effect in the presence of liposomes. In addition we determined dimensional characteristics of nanoparticles in a liposome suspension without and with sodium alginate and studied the influence of autoclave treatment on the nanoparticle size and cryoprotective effect of the complex. Autoclaving caused enlargement of a small-sized fraction of nanoparticles, supposedly, sodium alginate aggregates. Besides, the autoclaving did not demonstrate any influence on the cryoprotective efficiency of the mixture.

Butylated hydroxytoluene protects bull sperm surface protein-P25b in different extenders following cryopreservation.

Aim:The aim of this study was to investigate the effects of different concentration of butylated hydroxytoluene (BHT) on sperm membrane surface protein “P25b” from cryopreserved bull semen in either lecithin based Bioxcell® (BX) or two egg-yolk based extenders, tris-egg yolk (TEY), and citrate-egg yolk (CEY).Materials and Methods:Forty-five semen samples, 15 each were extended with either BX, TEY, or CEY extender which contained different concentrations (0.0 - control, 0.5, 1.0, 1.5, 2.0, and 3.0 mM/mL) of BHT. The extended semen samples were frozen at a concentration of 20×106/mL in 0.25 mL straws and stored in liquid nitrogen for 2weeks. The frozen samples were thereafter thawed, proteins extracted and analyzed for quantities of protein P25b through direct sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel densitometry. Peptides were confirmed by Western blotting (WB).Results:Results showed that supplementation of BHT improved (p<0.05) quantity of protein P25b at concentrations of 0.5mM/mL for BX and at 1.0 mM/mL for TEY and CE when compared with the controls and other treatments.Conclusion:BHT supplementation at 0.5 in BX and 1.0 mM/mL in TEY and CEY has protected bull sperm fertility marker protein P25b in frozen-thawed bull sperm.

Comparative analysis of various step-dilution techniques on the quality of frozen Limousin bull semen.

Background and Aim:Indonesia has two National Artificial Insemination centers and 17 Regional Artificial Insemination Centers. The frozen semen production techniques differed between the centers, including the type of diluent and semen dilution technique. The aim of the research was to compare the quality of frozen Limousin bull semen diluted using different techniques.Materials and Methods:Semen was collected from three sexually mature Limousin bulls using an artificial vagina. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen that had >70% motile sperm and <20% sperm abnormality was divided into three tubes and diluted with skim milk-egg yolk (SMEY) using three different dilution techniques: One-step dilution (100% SMEY with 8% glycerol) at room temperature ([RT] 20°C until 25°C) two-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol after 1 h stored at 5°C); and three-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol added twice at 1 h and 1.5 h after being stored at 5°C). The diluted semen was loaded into 0.25 mL mini straws, equilibrated, and frozen using a freezing machine. Sperm motility, viability, membranes, DNA integrity, and concentrations of malondialdehyde (MDA) and aspartate aminotransferase (AST) enzymes were evaluated after thawing.Results:The results showed that there were no significant differences in sperm motility and DNA integrity between dilutions (p>0.05). However, sperm viability and membrane intactness of one-step dilutions were higher than those of three-step dilutions. The concentrations of MDA and AST enzymes of sperm in one-step dilutions were lower than those of three-step dilutions (p<0.05).Conclusion:It was concluded that the one-step-dilution technique was better than three-step dilution for cryopreservation of Limousin bull semen.

Effect of Supplementation of Polyvinylpyrrolidone in Extender on Buffalo Semen Parameters

The current study was planned to evaluate the supplementation of Polyvinylpyrrolidone in extender on cryopreservation of Nili-Ravi buffalo bull semen. The semen samples were collected from Nili-Ravi buffalo (Bubalus bubalis) bull kept at SPU Qadirabad, District Sahiwal, Pakistan. Qualifying semen ejaculates having motility &gt;60%, volume &gt;5-6ml and concentration &gt;0.5 billion/ ml were diluted 50 × 106 motile sperm ml approximately at 37°C in Tris-citric acid extender supplemented with different concentrations of PVP (0.01, 0.05, 0.1mM). The extender without PVP was kept as control. Semen was stored at 4°C for a period of 2 h and kept at 4°C for 4h. Semen was filled in 0.5 ml French straws using suction pump at 4°C, plunged and stored in liquid nitrogen (-196°C). Semen straws were rewarmed at 37°C for 30 seconds and assessed for sperm motility, plasma membrane integrity (PMI), dead sperm percentage and the live sperm percentage. The data on the role of PVP on different parameters of semen quality were analyzed by using ANOVA and RCBD. Higher percentage (P&lt; 0.05) of sperm motility (66.1±7.51 and 59.4±10.72) and PMI (72.9±5.39 and 75.7±6.5) was observed in extenders having 0.05 mM and 0.1mM PVP compared to extenders having 1.5mM PVP and control. The percentage acrosomal integrity was observed greater (P&lt; 0.05) in extended semen containing 0.1mM (68.2±0.50) PVP compared to extenders having 0.01 and control.

Effect of various antioxidants and their combinations on bull semen cryopreservation

Our objective was to evaluate the effects of adding 50 mM trehalose (TRE), 1 mM cysteine (CYS), or 400 µg catalase (CAT), individually or in combinations, to a Tris-based semen extender and determine the effects on fresh, chilled-equilibrated, and frozen-thawed bull sperm. Ejaculates from three Holstein bulls (1-3 years old) were pooled, divided into six equal portions, and put into a Tris-based extender supplemented with no additive (control), TRE, CYS, CAT, TRE + CYS, or TRE + CYS + CAT. In chilled-equilibrated semen, motility was highest in CAT but lowest in the control (P < 0.05). In thawed semen, motility was lower (P < 0.05) in CAT than in all other groups, which were not different. Based on a hypoosmotic swelling test (HOS test), TRE, CYS, and their combinations had higher (P < 0.05) percentages of sperm with an intact plasma membrane than the control or CAT groups. The TRE group had the least (P < 0.05) morphologically abnormal sperm. Plasma membrane and acrosomal integrity (PMAI) and high mitochondrial membrane potential (HMMP) values were highest in TRE and TRE + CYS and were lower (P < 0.05) in the control. Furthermore, HMMP was positively correlated with PMAI and HOS tests, but negatively correlated with major + minor abnormalities (r = 0.66, 0.242, and -0.349). In conclusion, the addition of TRE, with or without CYS, to a Tris-based semen extender improved several aspects of the frozen-thawed quality of bull semen. However, addition of CAT, by itself or in combination with other additives, did not protect bull sperm against cryopreservation-induced defects.

Evaluation of Enrofloxacin for Use in Cryopreservation of Zebu Bull (Bos indicus) Semen.

Aim: The present study was designed to evaluate enrofloxacin (EN), a fluoroquinolone broad-spectrum antibacterial drug for use in cryopreservation of Zebu bull semen. Materials and Methods: Semen was collected from three Zebu bulls of the Sahiwal breed (Bos indicus) for 3 weeks (replicates) for each experiment. Experiment I: semen samples were cultured for isolation of bacteria and in vitro sensitivity tests against streptomycin, penicillin, and EN. Proteus spp., Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were isolated. P. aeruginosa was found to be resistant to streptomycin and penicillin (SP), while Proteus spp. was resistant to streptomycin. EN effectively inhibited the growth of all four bacteria in an in vitro sensitivity test. Experiment II: in vitro dose toxicity of EN (0-2000 μg/mL) for bull sperm was assessed in 2.9% sodium citrate buffer. EN up to a concentration of 1200 μg/mL was found to be safe for motility and viability of bull spermatozoa. Experiment III: semen was cryopreserved in a tris-citric acid extender containing either SP or EN (400 μg/mL) or without antibiotics as the control. Results: Post-thaw sperm motility was found to be higher in the extender containing EN and SP compared to control. Nevertheless, sperm viability was recorded higher (p < 0.05) with EN compared to SP and control. Sperm chromatin integrity did not differ (p > 0.05) in EN, SP, and controls. The total aerobic bacterial count was recorded as zero in the extender containing EN compared to SP and control. The fertility rate was higher (p < 0.05) with 400 μg/mL EN (59%) compared to SP (44%). Conclusions: It is concluded that EN is capable of controlling the bacterial load in frozen Zebu bull semen, resulting in a higher fertility rate under field conditions.

Nerve growth factor-β effects on post-thaw bull semen quality: Effects of nerve growth factor-β added to extenders for cryopreservation of electro-ejaculated and epididymal bull semen

Nerve growth factor-β (NGF) is a seminal plasma protein associated with improved sperm membrane integrity and motility in mammalian species. The objective of this study was to compare post-thaw semen quality from both ejaculated and pididymal-collected bull sperm incubated with purified NGF prior to cryopreservation. Semen was obtained from Angus × Simmental crossbred bulls (n = 10) collected by electroejaculation, followed by castration and epididymal sperm collections 3 days later. Semen samples were incubated with extender having 0 ng/mL (CONT), 0.5 ng/mL (LOW), 5 ng/mL (MED), or 50 ng/mL (HIGH) of purified NGF prior to cryopreservation. Sperm motility was assessed in each sample prior to treatment and cryopreservation and at post-thaw. Flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and chromatin stability (acridine orange). Values for post-thaw sperm motility and velocity variables were decreased, while linearity was increased in samples of the HIGH compared with CONT group (P < 0.01), but there were no differences in epididymal samples (P> 0.05). Samples from the HIGH group also had a lesser amplitude of lateral head displacement at 2.5 and 3 h post-thaw (P < 0.01). Post-thaw sperm viability, acrosome integrity, and DNA fragmentation index were not affected by NGF treatment in either ejaculated or epididymal sperm (P> 0.05). In conclusion, supplementation of freezing extender with NGF had minimal effects on post-thaw sperm quality in bulls. Results indicate NGF may have a function in preventing premature sperm hyperactivation in ejaculated, but not epididymal-collected spermatozoa. Fertility studies, both in vitro and in vivo, are warranted to ascertain the relevancy of these findings.

Enhancement of Extender Excellence of Frozen Bull Semen Using α-Tocopherol as an Antioxidant

Background: Addition of the antioxidant to extender media is the most appropriate attempt to reduce structural losses encounter during the process of cryopreservation. Hence semen excellence could be maintained for longer duration without adverse impact. Additionally antioxidants are not only capturing the reactive oxygen species but also improve the sperm quality indicators and fertility. Accordingly, current elucidation has been executed to explore the dose depended appraisal of varied concentration of α-tocopherol in Tris-based extender on frozen-thawed bull semen quality parameters for enhancement of bull semen cryopreservation in the subtropical ecosystem of Peshawar.Materials, Methods &amp; Results: Experiments were carried out on semen that has been collected from both Achai-an indigenous breed and Holstein Friesian (HF) - the exotic breed in artificial vagina maintained at 42°C from the experimental bulls of either breed and processed independently breed wise. Semen specimens with above 70% motility were evaluated separately breed-wise under same environmental condition. Standard procedure was adopted to extend the collected semen in the experimental extenders and frozen subsequently. After thawing, further Analysis of the frozen straws of semen was carried out for sperm excellence indicators that include motility, viability, acrosomal integrity and functional integrity of spermatozoa under the subtropical condition. Sperm viability and acrosomal integrity were determined by dual staining procedure i.e. Trypan-blue and Giemsa stains. The hypo-osmotic swelling (HOS) test was used to assess plasma membrane integrity. The current elucidation demonstrated that α-tocopherol 1.5 mg/mL supplemented in extender had significantly (P &lt; 0.05) increased sperm excellence gauge that includes motility, viability, acrosomal integrity and functional membrane integrity in both the breeds. On the other hand, the result further elucidated those concentrations higher or lower than 1.5-1 mg/mL α-tocopherol supplemented in the present study resulted in a lower semen functional attributes subsequent to freezing.Discussion: Biological samples were preserved by various methods such cryopreservation at low temperature to maintain them intact and facilitate downstream experiments. Antioxidants were associated with the chemical breakage of the substrate consequential from oxidation and counterbalance the free radicals thus diminish the damaging impacts to spermatozoa in cryopreservation. There is lack of data with respect to lipid peroxidation and cell reinforcement limit in cryopreserved semen, and cryopreservation is related with the generation of receptive oxygen species (ROS) which lead to lipid peroxidation (LPO) of sperm membranes, bringing about lost motility, viability and fertility of sperm. Recently, in the reproductive management of the dairy animals, an important focus of research is to overcome the deleterious impact associated with semen cryopreservation. Elucidation of differential dose depended expression of α-tocopherol in semen extender has been evident in this study. The results were corroborated with diverse mammalian species under varied ecological management setups. Furthermore the detrimental impact of high doses of α-tocopherol in the semen extender has been demonstrated. It was deduced from the current finding that addition of α-tocopherol via dose depended approach in Tri extender enhanced the cryosurvivability of both Achai indigenous breed and HF the exotic breed, under existing reproductive management in the local ecosystem of Peshawar.

Comparative effects of addition of superoxide dismutase and reduced glutathione on cryopreservation of Sahiwal bull semen

The present study aimed to investigate effects of superoxide dismutase (SOD) and reduced glutathione (GSH) on the quality of frozen-thawed semen of Sahiwal bulls. Semen was collected twice a week for 8 weeks by artificial vagina from six Sahiwal bulls, kept at the Semen Production Unit Qadirabad, Sahiwal-Pakistan. After gross and microscopic evaluation, qualifying semen ejaculates were divided into 10 equal aliquots and diluted in extenders enriched with no antioxidants (control); or supplemented with either SOD (50, 100 and 200 IU/mL), or GSH (0.5, 1 and 2 mM) or their combinations (50 IU/mL SOD and 0.5 mM GSH, 100 IU/mL SOD and 1 mM GSH and 200 IU/mL SOD and 2 mM GSH). Samples were then frozen and stored in liquid nitrogen at -196°C for 24 h. The following parameters were evaluated for semen quality: post-thawed sperm motility, viability, acrosome and membrane integrity. According to the results, sperm motility, viability, acrosome and membrane integrity were significantly (P&lt;0.05) higher in samples treated either with 100 IU/mL of SOD; 1 mM and 2 mM of GSH or 50 IU/mL of SOD plus 0.5 mM of GSH. In conclusion, semen quality might be improved by supplementing semen extenders with 100 IU/mL of SOD; 0.5 and 1 mM of GSH and combination of 50 IU/mL and 0.5 mM of SOD and GSH, respectively.