Cryopreservation Of Bull Semen Research Articles

Low Density Lipoprotein - important player in increasing cryoprotective efficiency of soybean lecithin-based bull semen extenders.

Currently, considering cryopreservation of bull semen, there is no clear consensus over the comparability of cryoprotective efficacy of extenders with soybean lecithin and those based on egg yolk. The objective of this study was to prove the use of Low Density Lipoprotein (LDL) extracted from hen-egg yolk as an enhancing factor for soybean lecithin-based extenders. In total, 35 ejaculates of (seven bulls x five ejaculates per bull) were collected and cryopreserved at a commercial insemination centre. The effect of the LDL addition to the extenders AndroMed® and Bioxcell® was tested in a 6% (v/v) concentration on spermatozoa after thawing. Modified extender composition effects were assessed on sperm functional parameters motility, plasma membrane, mitochondrial membrane potential and acrosomal integrity after thawing by CASA, flow cytometry and fluorescent microscopy, respectively. Based on kinematic parameters determined from CASA, k-means cluster analysis was used to classify individual spermatozoon into specific subpopulations (fast, medium fast and slow). A subpopulation of fast spermatozoa was increased in the presence of LDL in both selected extenders (P < 0.05). Moreover, the positive effect of LDL on sperm motility was confirmed by decreasing the percentage of sperm in slow subpopulation (P < 0.05). The effect of LDL addition on the incidence of spermatozoa with intact plasma membrane was not demonstrated in any case of extender used (P > 0.05). The percentage of sperm with intact acrosome was improved when LDL was added to Bioxcell® extender (P < 0.05). On the other hand, addition of LDL to AndroMed® extender improved mitochondrial intactness after thawing (P < 0.05). In conclusion, our results showed that adding LDL to selected soybean lecithin-based extenders considerably ameliorated the functional parameters of spermatozoa after thawing and thus this lipoprotein could represent an improving agent for soybean lecithin-based extender for bull semen cryopreservation.

The Effect of Green Tea Extract Supplementation in Bull Semen Cryopreservation

The Effect of Green Tea Extract Supplementation in Bull Semen Cryopreservation

Seasonal changes in the proteome of cryopreserved bull semen supernatant

Seasonal variability in cattle fertility may be attributed to male reproduction, including the quality of semen produced by males and its usefulness after cryopreservation. The exact molecular mechanisms responsible for such seasonal variation are not entirely known. The aim of our study was to evaluate the changes in the proteome of cryopreserved bull (Bos taurus) semen supernatant throughout the year. The quality of cryopreserved semen collected from the same Limousin bulls during spring, summer, autumn and winter (n = 5 in each group) was assessed by measuring sperm motility parameters using the CASA system and sperm viability and the level of sperm reactive oxygen species using flow cytometry. Two-dimensional difference in-gel electrophoresis analysis coupled with the MALDI-TOF mass spectrometry was used to detect seasonal changes in the proteome of cryopreserved bull semen supernatant. We found seasonal variability in the percentage of sperm motility (P < 0.05), which was the highest in autumn and winter and the lowest in spring and summer. There was an effect of season on sperm viability (P < 0.05), with the highest percentage of dead sperm recorded in summer. There was no effect of season on sperm levels of oxidation. Proteomic analysis identified 23 protein spots, representing 10 proteins that changed during the year (P < 0.05). Albumin, clusterin, spermadhesin 1 and platelet-activating factor acetylhydrolase precursor were most abundant during winter, which presumably reflected correct lipid composition and morphology of spermatozoa, as well as involvement in protection against premature capacitation. Moreover, osteopontin and nucleobindin 1 could also be connected to increased semen quality in this season. C-C motif chemokine 2 precursor, epididymal-specific lipocalin-5, peptidyl-prolyl cis-trans isomerase and seminal ribonuclease were most abundant during summer and probably reflected disturbances during spermatogenesis and sperm maturation. In summary, our study has shown that cryopreserved bull semen quality parameters were higher in winter than in summer. The identification of proteins connected to the variability of semen quality during the year have provided new insight into understanding the mechanisms underlying the seasonality of cattle breeding.

The combination of CASA kinetic parameters and fluorescein staining as a fertility tool in cryopreserved bull semen

The combination of CASA kinetic parameters and fluorescein staining as a fertility tool in cryopreserved bull semen

Vital Stain in Milk-Based Medium to Evaluate Sperm Quality

Assisted reproductive technology plays a key role in bovine reproduction in herds. Different media, including the milk-based medium, are used for bull semen cryopreservation. The objective of the survey was to develop a method for assessing sperm motility in frozen-thawed semen of the bull sires with the use of the vital stains added to the milk-based medium. It has been proven that vital staining with the use of Hoechst 33342 Dye in the milk-based medium is the most efficient in order to assess the motility of the frozen-thawed semen collected from the bull sires. The current method for semen detection can yield 100% accurate results.

Effect of semen processing methods on lumpy skin disease virus status in cryopreserved bull semen

Lumpy skin disease is an economically important disease of cattle, caused by the lumpy skin disease virus (LSDV; Capripoxvirus). It has a variable clinical appearance but, in severely affected animals, is associated with extensive skin damage, pneumonia and death. The LSDV can be found in the semen of infected bulls for prolonged periods of time, from where it can be transmitted by mating or artificial insemination and cause clinical disease in heifers and cows. In this study, an ejaculate was collected from a LSDV seronegative bull and confirmed free from LSDV DNA by PCR. The ejaculate was split into a control sample (C), a sample spiked with a 4 log TCID50 dose of an LSDV isolate (HD) and a 103 dilution of the virus suspension (ND) and frozen routinely. Two straws from each of the different semen treatment groups (HD, ND and C) were subsequently thawed and subjected to swim-up, single layer centrifugation, Percoll® density gradient and a Percoll® density gradient with added trypsin. For one set of straws, semen quality variables were recorded, and viral DNA status determined using PCR; the other set was used for positive staining electron microscopy. Samples determined to be positive for LSDV DNA by PCR were then subjected to virus isolation (VI). Complete elimination of LSDV from semen did not occur with use of any of the processing methods. Trypsin did reduce the viral load, and eliminated LSDV from the ND sample, but severely negatively influenced semen quality. The LSDV virions, as assessed by electron microscopy, were associated with the sperm plasma membrane. Further investigation is needed to establish the efficacy of immuno-extenders for rendering semen free from LSDV.

Supplementation of quercetin for advanced DNA integrity in bull semen cryopreservation.

The aim of this study was to identify the effects of adding quercetin (Q) to Tris extender in order to identify levels of oxidative stress in bull sperm after freeze thawing. Ejaculates were collected via artificial vagina from Holstein bulls. Semen was divided into five tools and diluted to a final concentration of 15×106 spermatozoa/ml with the Tris extender containing Q (25, 50, 100 and 200μg/ml) and no additive (control; C). All examples were equilibrated at 4°C during 4hr then were loaded into 0.25-ml straws and frozen using a controlled rate. Sperm motility and motility characteristics were determined using the computer-assisted semen analyser. Sperm membrane integrity was assessed using the hypoosmotic swelling test. Sperm chromatin integrity was investigated using the single cell gel electrophoresis. Total antioxidant capacities were performed colorimetrically. Q supplementation used as an antioxidant did not produce better results in the proportion of sperm progressive and total motility, plasma membrane integrity and sperm abnormalities. Q supplementation exhibited the favourable tail length, tail DNA and tail moment. In conclusion, when whole parameters are considered, Q25 can be added to the Tris extender due to its positive effect on sperm DNA integrity and no adverse effect on the progressive and total motilities of sperm.

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.

Polyphenolics from Albizia harveyi Exhibit Antioxidant Activities and Counteract Oxidative Damage and Ultra-Structural Changes of Cryopreserved Bull Semen.

Albizia harveyi is a tropical deciduous tree, found across South and Eastern Africa and widely used in traditional medicine. The leaf extract ameliorated the damaging effects of the frozen-thawing process in cryopreserved bull semen. In a dose-dependent pattern, sperm motility, viability, and membrane integrity were improved compared to the untreated control. Furthermore, the extract increased the percentage of viable sperm cells and reduced the percentages of early apoptotic and apoptotic sperm cells as well as the damage in sperm ultra-structure. These activities are in agreement with the robust antioxidant properties in vitro and in the seminal fluid as observed in the total antioxidant capacity and the lipid peroxidation parameter malondialdehyde. LC-MS yielded 35 compounds. The extract was dominated by quercetin-O-galloyl-hexoside and quercetin-O-pentoside, along with other flavonoid glycosides. The polyphenols are probably responsible for the observed activities. In conclusion, the current findings show that A. harveyi leaves are rich in bioactive polyphenols with functional properties, validating its traditional use.

Alpha-tocopherol improves frozen-thawed sperm quality by reducing hydrogen peroxide during cryopreservation of bull semen

This study was conducted to investigate the effects of different levels of α-tocopherol in cryopreservation media on the bull sperm quality after thawing. Semen samples were collected from six Holstein bulls using artificial vagina twice a week. Semen samples were pooled to eliminate individual differences and then divided into four equal parts for freezing with extenders containing different concentrations of α-tocopherol according to experimental groups as follows: 0 (control), 1.2 mM (E1), 2.4 mM (E2) and 4.8 mM (E4). Motion characteristics, viability, plasma membrane functionality, lipid peroxidation and H2O2 status were determined after thawing. Results showed that malondialdehyde (MDA) concentration was significantly lower in E4 (6.1±0.6 nmol/mL) than E1 and E2 extenders (8.1±0.6 nmol/mL and 8±0.6 nmol/mL, respectively). Also, the lowest significant concentration of H2O2 was observed in E4 (3.2±0.13 nmol/mL) compared to E1 (4 ±0.1 nmol/mL), E2 (5.3± 0.1 nmol/mL) and control (6.7±0.1 nmol/mL). Moreover, E2 and E4 produced the highest significant motility (74.2±1.6%, 75.9±1.6%), viability (78.2±1.8%, 76.1±1.8%) and membrane functionality (73±1.6%, 70.5±1.6%) compared to other groups. It can be concluded that α-tocopherol at the concentration of 4.8 mM can be an efficient antioxidant additive in Bioxcell extender for cryopreservation of bull semen.

Effect of cholesterol loaded cyclodextrin supplementation on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa

The beneficial effects of cholesterol loaded cyclodextrin (CLC) addition were evaluated in cryopreserved bull semen. Forty ejaculates were collected from Hariana bulls (n = 4), pooled and divided into 4 aliquots. All the aliquots were initially diluted in to egg yolk tris citrate and supplemented with CLC @ 0.5 mg (Group-II), 1.0 mg (G-III) and 2.0 mg (G-IV) CLC/120 × 106 spermatozoa or without CLC (G-I) that served as control. Extended semen was cryopreserved at −196 °C for 24 h. Seminal attributes like motility, viability, cryocapacitation like changes, tyrosine phosphorylation, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA integrity were evaluated after equilibration and thawing. Results showed a significant increase in the motility, viability and acrosome intact spermatozoa in Group II as compared to other three groups. Further, the proportion of spermatozoa showing capacitation and acrosome reaction was also decreased (P < 0.05) significantly in Group II as compared to Group I, III, and IV. Immunoblot demonstrated a 32 kDa (p32) protein showing differential variation in the band intensity in all the four groups being lower in Group II. Further, the immunolocalization study revealed positive immune reactivity for tyrosine phosphorylated proteins at middle piece and neck (high fluorescence), post-acrosomal region (medium fluorescence), and principal piece (low fluorescence) of spermatozoa. Addition of CLC significantly increased (P < 0.05) the percentage of spermatozoa showing high transmembrane mitochondrial potential, and also, CLC @ 0.5 mg/120 × 106 in semen extender significantly decreased (P < 0.05) spermatozoa showing fragmented DNA after thawing as compared to control. Results of the present study indicate beneficial effects of CLC supplementation on cryodamage of spermatozoa by reducing the cryocapacitation and apoptosis like changes.

Cryopreservation of bull semen is associated with carbonylation of sperm proteins

Artificial insemination with cryopreserved semen enables affordable, large-scale dissemination of gametes with superior genetics. However, cryopreservation can cause functional and structural damage to spermatozoa that is associated with reactive oxygen species (ROS) production, impairment of sperm motility and decreased fertilizing potential, but little attention has been paid to protein changes. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of bull spermatozoa proteins triggered by the cryopreservation process. Flow cytometry and computer-assisted sperm analysis were used to evaluate changes in viability, ROS level and motility of spermatozoa. Western blotting, in conjunction with two-dimensional electrophoresis (2D-oxyblot) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight spectrometry, was employed to identify and quantify the specifically carbonylated spermatozoa proteins. Cryopreservation decreased motility and viability but increased the number of ROS-positive cells. We identified 11 proteins (ropporin-1, outer dense fiber protein 2, glutathione S-transferase, triosephosphate isomerase, capping protein beta 3 isoform, actin-related protein M1, actin-related protein T2, NADH dehydrogenase, isocitrate dehydrogenase, cilia- and flagella-associated protein 161, phosphatidylethanolamine-binding protein 4) showing differences in protein carbonylation in response to cryopreservation. The identified proteins are associated with cytoskeleton and flagella organization, detoxification and energy metabolism. Moreover, almost all of the identified carbonylated proteins are involved in capacitation. Our results indicate for the first time that cryopreservation induces oxidation of selected sperm proteins via carbonylation. We suggest that carbonylation of sperm proteins could be a direct result of oxidative stress and potentially lead to disturbances of capacitation-involved proteins or could indicate cryopreservation-induced premature capacitation.

The combination of kinetic and flow cytometric semen parameters as a tool to predict fertility in cryopreserved bull semen

Within recent years, there has been growing interest in the prediction of bull fertility through in vitro assessment of semen quality. A model for fertility prediction based on early evaluation of semen quality parameters, to exclude sires with potentially low fertility from breeding programs, would therefore be useful. The aim of the present study was to identify the most suitable parameters that would provide reliable prediction of fertility. Frozen semen from 18 Italian Holstein-Friesian proven bulls was analyzed using computer-assisted semen analysis (CASA) (motility and kinetic parameters) and flow cytometry (FCM) (viability, acrosomal integrity, mitochondrial function, lipid peroxidation, plasma membrane stability and DNA integrity). Bulls were divided into two groups (low and high fertility) based on the estimated relative conception rate (ERCR). Significant differences were found between fertility groups for total motility, active cells, straightness, linearity, viability and percentage of DNA fragmented sperm. Correlations were observed between ERCR and some kinetic parameters, and membrane instability and some DNA integrity indicators. In order to define a model with high relation between semen quality parameters and ERCR, backward stepwise multiple regression analysis was applied. Thus, we obtained a prediction model that explained almost half (R2=0.47, P<0.05) of the variation in the conception rate and included nine variables: five kinetic parameters measured by CASA (total motility, active cells, beat cross frequency, curvilinear velocity and amplitude of lateral head displacement) and four parameters related to DNA integrity evaluated by FCM (degree of chromatin structure abnormality Alpha-T, extent of chromatin structure abnormality (Alpha-T standard deviation), percentage of DNA fragmented sperm and percentage of sperm with high green fluorescence representative of immature cells). A significant relationship (R2=0.84, P<0.05) was observed between real and predicted fertility. Once the accuracy of fertility prediction has been confirmed, the model developed in the present study could be used by artificial insemination centers for bull selection or for elimination of poor fertility ejaculates.

Effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen

This study was designed to investigate the effects of an equilibration period up to 96 hours and three extenders (AndroMed, OPTIXcell, and Triladyl) on the quality of cryopreserved bull semen and to evaluate, whether an extension of the equilibration time to 72 hours does affect fertility in the field. One ejaculate of 17 bulls was collected and divided into three equal aliquots and diluted, respectively, with the three extenders. Each aliquot was again divided into five parts and equilibrated for 4, 24, 48, 72, and 96 hours before freezing in an automatic freezer. Sperm motility, plasma membrane and acrosome integrity (PMAI), and DNA fragmentation index (% DFI) were measured during equilibration. In addition to the parameters measured during equilibration, the percentage of viable sperm cells with high mitochondrial membrane potential (HMMP) was measured immediately after thawing, and after 3 hours of incubation at 37 °C. Sperm motility was assessed using CASA, and PMAI, HMMP, and % DFI were measured using flow cytometry. Equilibration time did affect all parameters before freezing (P < 0.01), and also the extender affected all parameters except HMMP (P < 0.05). After thawing, all parameters except HMMP immediately after thawing were influenced by the equilibration period (P < 0.001), whereas all parameters except % DFI immediately after thawing were influenced by the extender (P < 0.001). The changes of semen characteristics during 3 hours of incubation were also dependent on the equilibration time and the extender used in all parameters (P < 0.01). In the field study, semen of nine bulls was collected thrice weekly, processed using Triladyl egg yolk extender, and frozen in 0.25 mL straws with 15 × 106 spermatozoa per straw. In total, the nonreturn rates on Day 90 after insemination (NRR90) of 263,816 inseminations in two periods were evaluated. Whereas semen collected on Mondays and Wednesdays was equilibrated for 24 hours in both periods, semen collected on Fridays was equilibrated for 4 hours in period one and equilibrated for 72 hours in period 2. No differences in NRR90 could be found (P > 0.05). In conclusion, extension of the equilibration time from 4 hours to 24–72 hours can improve motility and viability of cryopreserved semen after thawing. The extent of improvement in semen quality is dependent on the extender used. Prolongation of the equilibration period from 4 hours to 72 hours had no effect on fertility in the field.

Effect of seminal plasma removal before cryopreservation of bovine semen obtained by electroejaculation on semen quality and in vitro fertility

Cryopreservation of bull semen is a common biotechnology procedure in cattle breeding. However, when the ejaculate is obtained by electroejaculation, wide variation is observed in the sperm/seminal plasma (SP) ratio that can affect the freezability of semen in this species. The removal of SP may improve the quality of frozen bull semen. The objective of this study was to evaluate the effect of SP removal from the ejaculate on the cryopreservation of semen from 38 Nellore bulls collected by electroejaculation. After collection, the ejaculate was divided into three aliquots: (1) control (N) diluted to a concentration of 60 × 106 spermatozoa/mL and frozen with SP; (2) centrifugation (C) at ×600g for 10 minutes and the pellet resuspended and frozen at the same concentration as N; and (3) filtration (F) through SpermFilter and sperm recovered and frozen at the same concentration as N. After thawing, sperm kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, oxidative stress, and in vitro fertility were evaluated. Statistical analysis was performed using the SAS 9.2 package, and differences were considered significant when P < 0.05. Higher average path velocity and straight-line velocity were observed in the groups submitted to SP removal compared to the control group (P < 0.01). In contrast, filtered samples exhibited higher beat cross frequency, straightness, and linearity compared to the other groups. Plasma membrane integrity was reduced when SP was removed, but lower oxidative stress was observed in groups C and F (34.91 ± 2.95% and 31.63 ± 2.95%, respectively) compared to group N (57.39 ± 2.95%). However, the percentage of hatched blastocysts was similar in the N and F groups (21.22 ± 1.05% and 24.00 ± 1.05%, respectively) and higher compared to group C (18.83 ± 1.05%). In conclusion, removal of SP by centrifugation for bull semen freezing reduced the rate of in vitro–produced embryos, whereas filtration of prefrozen semen was found to be an efficient alternative in terms of semen freezability and in vitro production of bovine embryos.

Evaluation of quail and turkey egg yolk for cryopreservation of Nili-Ravi buffalo bull semen

Egg yolk is used as a cryoprotectant for semen in different mammalian species including buffalo. Egg yolk from different sources may affect freezability of buffalo bull semen. Quail egg yolk (QEY) and turkey egg yolk (TEY) in Tris-citric acid extender was evaluated for post-thaw quality and in vivo fertility rate of cryopreserved buffalo bull semen. Ejaculates were collected on weekly basis from six Nili-Ravi buffalo bulls (12 ejaculates/bull) for a period of 6 weeks and diluted at 37 °C with tris-citric egg yolk extender (50 × 106 motile spermatozoa mL−1) containing different levels of QEY or TEY (5%, 10%, 15%, and 20%) or 20% chicken egg yolk (CEY; controls) and cryopreserved. Percent post-thaw sperm motility (48.3 ± 3.8), plasma membrane integrity (67.9 ± 5.3), live/dead ratio (68.2 ± 5.0), and viability (50.5 ± 3.7) were recorded higher (P < 0.05) in extender containing 5% QEY compared with control. However, TEY at 10% in extender improved (P < 0.05) the post-thaw sperm motility (57.5 ± 5.2), plasma membrane integrity (53.5 ± 4.5), livability (75.3 ± 6.0), and viability (73.5 ± 6.5) compared with higher concentrations of TEY and controls (20% CEY). The chromatin damage (2.0 ± 0.9) and intracellular enzymes, glutamic oxaloacetic transaminase (24.8 ± 3.5) and lactic dehydrogenase (77.7 ± 4.5), release were lower (P < 0.05) in extender containing 10% TEY compared with the controls. Invivo fertility was compared after artificial insemination with semen from two buffalo bulls that was cryopreserved in extenders containing 5% QEY, 10% TEY, or 20% CEY. A total of 600 inseminations (200 inseminations per extender) were recorded; the overall fertility rate was significantly higher (P < 0.05) with semen cryopreserved in extender containing 5% QEY (57.5 vs. 42%) and 10% TEY (57.5 vs. 42%). compared with 20% chiken egg yolk. In conclusion, QEY at 5% and TEY at 10% offers advantages over 20% CEY in terms of in vitro post-thaw semen quality and in vivo fertility of buffalo.

A 31-kDa seminal plasma heparin–binding protein reduces cold shock stress during cryopreservation of cross-bred cattle bull semen

In the present study, a 31-kDa protein, purified from cattle bull seminal plasma heparin–binding proteins (SP-HBP), was characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass spectrometry. Raw semen of six cross-bred bulls was treated with 31-kDa HBP before cryopreservation to observe its effect on motility, viability, hypo-osmotic swelling test, acrosome integrity, in vitro capacitation/acrosome reaction, and oxidative stress at pre-freeze and frozen–thawed phases of cryopreservation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of 31-kDa protein eluted and purified from SP-HBP (separated on acrylamide gels) resulted in a single band of 40 kDa. In matrix-assisted laser desorption/ionization–time of flight analysis, 12 peptides were identified with matching significantly (P < 0.05) to interlukin-6 of bovine with a top score of 55. Addition of 25 μg/mL of fluorescein isothiocyanate–conjugated 31-kDa protein to raw semen and incubation at 37 °C for 20 minutes before cryopreservation resulted in its binding mainly to head region. Treatment of semen with 31-kDa HBP resulted in a significant (P < 0.05) average increase of 9.2%, 6.8%, and 11.7% and 5.5%, 6.5%, and 11.0% in motile, viable, hypo-osmotic swelling–responsive spermatozoa in six bulls at pre-freeze and frozen–thawed phases of cryopreservation, respectively. Percentage of spermatozoa with intact acrosomes nonsignificantly enhanced in the semen treated with 31-kDa HBP at both phases of cryopreservation. An average nonsignificant increase of 3.1% in in vitro capacitated and acrosome-reacted spermatozoa was obtained in semen supplemented with 31-kDa HBP. Addition of 31-kDa HBP also nonsignificantly reduced Malonadialdehyde (MDA) level by 10.7 and 19.3 μM/109 spermatozoa in prefrozen and frozen–thawed semen, respectively. The results obtained here indicate to conclude that treatment of cross-bred cattle bull semen with 31-kDa HBP protects the spermatozoa from cold shock effect by coating the sperm surface.

Effects of the platelet-activating factor (PAF) on selected quality parameters of cryopreserved bull semen (AI) with reduced sperm motility.

The aim of the study was to determine the effects of platelet-activating factor (PAF) on selected quality parameters of cryopreserved bull semen with reduced sperm motility used for artificial insemination. The aim of experiment 1 was to identify the optimal concentration of the phospholipid able to preserve sperm viability. Cryopreserved semen was treated with different PAF concentrations: 1×10(-5) M, 1×10(-6) M, 1×10(-7) M, 1×10(-8) M and 1×10(-9) M. The experiment demonstrated that PAF at concentration 1×10(-9) M increased most the sperm viability parameters (motility parameters, plasma membrane integrity and mitochondrial function) after 120 min of incubation of thawed semen at 37°C. Cryopreserved bull semen with reduced sperm motility (below 70%) was supplemented with PAF in a concentration of 1×10(-9) M. A statistically significant increase in sperm motility, percentage of linear motile spermatozoa and VSL value was observed after 120 min incubation of sperm with 1×10(-9) M PAF. Sperm supplementation with PAF also had positive effects on plasma membrane integrity and percentage of spermatozoa with preserved mitochondrial transmembrane potential, but the differences were not statistically significant. The results indicated positive effects of PAF supplementation at a concentration of 1×10(-9) M on the selected sperm quality parameters in cryopreserved bull semen with reduced motility.

Impact of cryopreservation on bull () semen proteome.

Cryopreservation of bull spermatozoa is a well-established technique, allowing artificial insemination of cattle on a commercial scale. However, the extent of proteome changes in seminal plasma and spermatozoa during cryopreservation are not yet fully known. The objective of this study was to compare the proteomes of fresh, equilibrated, and cryopreserved bull semen (spermatozoa and seminal plasma) to establish the changes in semen proteins during the cryopreservation process. Semen was collected from 6 mature Holstein Friesian bulls. After sample processing, comparative analysis and identification of proteins was performed using 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. Analysis of spermatozoa extracts revealed that 25 identified protein spots, representing 16 proteins, underwent significant ( < 0.05) changes in abundance due to equilibration and cryopreservation. Eighteen protein spots decreased in abundance, 5 protein spots increased in abundance, and 2 protein spots showed different, specific patterns of abundance changes. Analysis of seminal fluid containing seminal plasma showed that 6 identified protein spots, representing 4 proteins, underwent significant ( < 0.05) changes in abundance due to equilibration and cryopreservation. Two protein spots increased in abundance and 4 decreased in abundance. Semen extending and equilibration seems to be responsible for a significant portion of the proteome changes related to cryopreservation technology. Most sperm proteins affected by equilibration and cryopreservation are membrane bound, and loss of those proteins may reduce natural spermatozoa coating. Further research is needed to unravel the mechanisms of the particular protein changes described in this study and establish the relationship between those changes and sperm quality.

Seminal Plasma Heparin Binding Proteins Improve Semen Quality by Reducing Oxidative Stress during Cryopreservation of Cattle Bull Semen.

Heparin binding proteins (HBPs) are produced by accessory glands. These are secreted into the seminal fluid, bind to the spermatozoa at the time of ejaculation, favour capacitation, acrosome reaction, and alter the immune system response toward the sperm. The present study was conducted with an objective to assess the effect of purified seminal plasma-HBPs (SP-HBPs) on cross bred cattle bull sperm attributes during two phases of cryopreservation: Pre freezing and freezing-thawing. SP-HBPs were purified from pooled seminal plasma by heparin affinity chromatography. Three doses of SP-HBPs i.e. 10, 20, 40 μg/mL semen were standardized to find out the optimum dose and 20 μg/mL was found to be an optimum dose. Semen as such and treated with SP-HBPs was diluted with sodium citrate-egg yolk diluter and cryopreserved as per the standard protocol. Sperm parameters i.e. motility, viability, Hypo-osmotic swelling test (HOST), acrosome damage, in vitro capacitation and lipid peroxidation were evaluated in SP-HBP treated and untreated (control) semen at both phases of cryopreservation. A considerable variation in percent sperm motility, viability, membrane integrity (HOST), acrosome damage, acrosome reaction and lipid peroxidation was observed at both phases among the bulls irrespective of the treatment. Incubation of neat semen with 20 μg/mL SP-HBP before processing for cryopreservation enhanced the average motility, viability, membrane integrity by 7.2%, 1.5%, 7.9%, and 5.6%, 6.6%, 7.4% in pre-frozen and frozen-thawed semen in comparison to control. There was also an average increase of 4.1%/3.9% in in vitro capacitation and acrosome reaction in SP-HBPs-treated frozen-thawed semen as compared to control. However, binding of SP-HBPs to the sperm declined acrosome damage and lipid peroxidation by 1.3%/4.1% and 22.1/32.7 μM/109 spermatozoa in SP-HBP treated pre-frozen/frozen-thawed semen as compared to control, respectively. Significant (p<0.05) effects were observed only in motility, HOST and in vitro acrosome reaction. It can be concluded that treatment of neat semen with SP-HBPs before cryopreservation minimized the cryoinjury by decreasing the generation of reactive oxygen species.