Cryopreservation Of Bull Semen Research Articles

Long-term cryopreservation of bull semen as a main strategy for the ex situ conservation of endangered Polish Red cattle

Abstract Regular verification of the quality of cryopreserved semen derived from native cattle is one of the tasks performed at the bank as recommended by the Food and Agriculture Organization. The purpose of the present study was to evaluate the quality of semen from PR bulls stored for 40, 50 and 60 years in the BMB using standard evaluation parameters such as sperm motility as well as structural-functional parameters such as plasma membrane integrity, transmembrane mitochondrial potential and sperm chromatin damage. Semen pellets from 27 PR bulls (3 ejaculates/bull) were tested. The data were analysed by one-way and two-way ANOVA, and the significance of the difference (P≤ 0.01) between the means was determined using Duncan’s test. Our study results revealed that the long-term storage of semen had no effect on sperm characteristics after thawing. However, statistically significant differences (P≤0.01) in sperm plasma membrane integrity and transmembrane mitochondrial potential, following storage in liquid nitrogen were noted between bulls at all time points. However, there were no significant differences (P>0.01) in sperm chromatin damage between breeds or between different storage times, and the degree of DNA fragmentation ranged from 0.4 to 0.8%.

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 the addition of soursop juice (Annona muricata L., 1753) to egg yolk citrate extender on the quality of bull semen

The present study was carried out to investigate the effect of the addition of soursop (Annona muricata L., 1753) juice to the citrate-egg yolk extender (EYC) on the quality of cryopreserved bull semen. After pre-evaluation, the semen was distributed in five extenders T1, T2, T3, T4 and T5 containing respectively 0% soursop juice (SJ) (T1), 25% SJ (T2), 50% SJ (T3), 75% SJ (T4), 100% SJ (T5) and microscopic evaluation was performed daily to assess plasma membrane integrity (PMI), progressive motility of spermatozoa (MP) and sperm pH until all sperm were dead. The results indicate a significant increase in spermatozoa PMI and MP (p<0.05) in the case of treatment with 50% SJ (T3). Spermatozoa showed a motility of 50.78% 9 days later in the citrate-egg yolk extender containing 50% SJ (T3), whereas at day 9, spermatozoa in the other extenders had a progressive motility of less than 50%. Similar results were obtained for PMI with 50.78% of spermatozoa having an intact plasma membrane 9 days later in the citrate-egg yolk extender containing 50% SJ whereas the PMI of cells in the other stents was less than 50%. However, the pH of the extender containing 50% SJ was closest to 7 during the experimental period. The results of this experiment suggest that the addition of 50% soursop juice (T3) to the citrate-egg yolk extender could be adopted as an acceptable alternative to traditional egg yolk-based extenders for the cryopreservation of bull semen.

Bull sperm cryopreservation: An overview on the current status and future perspectives

Cryopreservation refers to freezing cells or tissues at extremely low temperatures, allowing them to be stored for extended periods while maintaining viability. Cryopreserved bull semen has become an essential tool in cattle breeding programs and commercial cattle production systems. This review provides a detailed analysis of the current methods and challenges in preserving bull sperm using cryopreservation. We explore the effects of cryopreservation on sperm cells, the role of different cryoprotectants, as well as the progress made in the analysis of bull semen. It also highlights the impact of the freezing process on sperm morphology and functionality, emphasizing the importance of optimizing cryopreservation techniques to maintain sperm fertility and viability. The article underscores the significance of cryopreservation technology in cattle genetics and breeding and suggests future research to enhance cryopreservation techniques.

CASA Based Assessment of Kangayam bull semen processed with different extenders and Conception rate following AI

Semen extenders are the essential components of cryopreservation of semen. Identification of suitable and ready to use semen extender is the need of hour for the rapid propagation of Kangayam cows through artificial insemination. Kangayam bull, aged 5 years was utilized for the study. The semen was collected twice in a week and each time two ejaculates were collected. A total of 24 ejaculates were utilized for this study. After assessing the general macroscopical examination the semen was subjected to computer aided semen analysis (CASA) for assessing the kinematic parameters in fresh diluted semen, pre freeze and post thawed such as sperm motility, progressive individual motility, VAP, VSL, ALH and LIN were analyzed using CASA. Among all the three extenders, the sperm kinematic parameters during fresh diluted, pre freeze and post thawed semen stages were higher in group III (liposome based extender) followed by group I (TEYG) and in group II (soya bean lecithin based extender). Among the three extenders used, the components of liposome based extender was better and it was able to maintain the structural integrity, plasma membrane integrity and motility in the sperms of the frozen thawed semen than the other two extenders (TEYG and soya bean lecithin based extenders). Further the fertility rate was evaluated by the in vivo insemination of 60 Kangayam cows after 12 hours of onset of estrum. The fertility rate observed was 45.00, 40.00 and 65.00 per cent in groups I, II and III, respectively. The overall conception rate recorded in Kangayam cow was 50.00 per cent. Based on the findings, it was concluded that liposome based extender may be used for cryopreservation of Kangayam bull semen in order to maximize the fertility rate.

Correlation between in vitro sperm kinetic, oxidative stress assessments and field fertility of cryopreserved bull semen

This study assessed kinetic parameters and oxidative stress in bull sperm after post-thaw (PT) or after sperm selection by Percoll™ gradient, and thermo resistance test (SS + TRT) to identify useful indicators of field fertility. For the experiment, commercial doses of frozen semen were obtained from six Aberdeen Angus bulls. Three of the bulls were classified as high fertility and three as low fertility according to the IFert™ index provided by the international breeding company CRV Lagoa. Pooled semen samples were distributed between two treatment groups for analysis: post-thaw (PT) or sperm selection (SS) (Percoll™) and thermal resistance test (SS + TRT). The samples were evaluated using sperm kinetics (CASA) (motility %, progressive motility %, VCL µm/s, VSL µm/s, VAP µm/s, LIN %, STR % and WOB%), production of reactive oxygen species (ROS), lipid peroxidation, superoxide dismutase (SOD) enzyme activity and total antioxidant capacity. Data were analyzed using Two-Way ANOVA, considering the fertility index, the treatment used in the samples as effects, and the interaction between these factors. When a significant effect was observed, the values were compared using the Bonferroni test. A Pearson Correlation analysis was performed between the fertility indices and the sperm parameters analyzed in vitro, to evaluate the relationship between sperm quality and the fecundity rate obtained by the bulls. Sperm kinetic parameters, including total motility, progressive motile, and beat cross-frequency, were higher in low fertility compared to high fertility bulls (P < 0.05). However, curvilinear velocity was greater in high fertility bulls followed by SS + TRT. Straight-line velocity, average path velocity, linearity, and beat cross-frequency beat were higher in high fertility bulls after SS + TRT. Reactive oxygen species was correlated with fertility after SS. In addition, there was a decrease in lipid peroxidation was observed only in high fertility bulls. However, lipid peroxidation and high fertility were correlated after PT and SS + TRT. The combination of in vitro sperm kinetic parameters predicted in vivo fertility more accurately than individual kinetic parameters. The lipid peroxidation of sperm is an important indicator of fertility in bulls. High fertility bulls appeared to be more susceptible to lipid peroxidation, which was only reduced in high fertility bulls, suggesting that their sperm can repair the damage induced by oxidative stress.

Tinospora cordifolia modulates the seminal parameters, leakage of intracellular enzymes and seminal antioxidants in equilibrated and cryopreserved semen of Sahiwal bulls.

The present study was undertaken to assess the effects of stem extract of Tinospora cordifolia (Giloy or Guduchi) in the semen extender on seminal parameters, leakage of intracellular enzymes and antioxidants in semen of Sahiwal bull. A total of 48 ejaculates from four bulls were selected for the study. Spermatozoa of 25×106 were incubated in 100, 300 and 500 μg of stem extract of Guduchi as Gr II, III and IV, respectively and pre-freeze and post-thaw semen samples were analysed for seminal parameters (motility, viability, total sperm abnormality; TSA, plasma membrane integrity; PMI, and acrosomal integrity; AcI), intracellular enzymes (aspartate aminotransferase; AST and lactate dehydrogenase; LDH) and seminal antioxidants (superoxide dismutase; SOD and catalase) in comparison with an untreated control group (Gr I). The results revealed that stem extract treated semen had significantly (p<0.05) higher motility, viability, PMI, AcI, SOD and catalase and had significantly (p<0.05) lower TSA, AST and LDH compared to those in untreated control group at pre-freeze and post-thaw stages. Semen treated with 100 μg stem extract /25×106 spermatozoa had significantly (p<0.05) higher motility, viability, PMI, AcI, SOD and catalase and had significantly (p<0.05) lower TSA, AST and LDH compared to those in control, 300, and 500 μg treated groups at pre-freeze and post-thaw stages. Further, these seminal parameters and antioxidants were showing decreasing trend and TSA and leakage of intra-cellular enzymes were showing increasing trend from Gr II to Gr IV at pre-freeze and post-thaw stages. Thus, 100 μg/25×106 spermatozoa were optimum or suitable dose for cryopreservation of Sahiwal bull semen. The study concluded that T. cordifolia stem extract 100 μg/25×106 spermatozoa in the semen extender can be effectively utilized to reduce the oxidative stress and improve the pre-freeze and post-thaw seminal parameters in Sahiwal bull. However, further studies on effects of different concentrations of stem extract on in-vitro or in-vivo fertility trials are to be conducted to assess the impact of the stem extract supplementation in the semen extender on field pregnancy outcome in bovine species.

Glutathione and selenium nanoparticles have a synergistic protective effect during cryopreservation of bull semen.

In the present study, the synergistic protective effect of co-supplementation of glutathione (GSH) with selenium nanoparticles (SeNPs) on the cryopreservation efficiency of bull semen was analyzed. After collection, the ejaculates of Holstein bulls were subsequently diluted with a Tris extender buffer supplemented with different concentrations of SeNPs (0, 1, 2, and 4 μg/ml), followed by semen equilibration at 4°C and assessment of sperm viability and motility. Subsequently, the ejaculates of Holstein bulls were pooled, split into four equal groups, and diluted with a Tris extender buffer supplemented with basic extender (negative control group, NC group), 2 μg/ml SeNPs (SeNPs group), 4 mM GSH (GSH group), and 4 mM GSH plus 2 μg/ml SeNPs (GSH + SeNPs group). After cryopreservation, motility, viability, mitochondrial activity, plasma membrane integrity, acrosome integrity, concentration of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), and ability of frozen-thawed sperm cells to support in vitro embryonic development were evaluated. No side effect of SeNPs concentrations applied in the current study on the motility and viability of equilibrated bull spermatozoa was found. Meanwhile, supplementation of SeNPs significantly promoted the motility and viability of equilibrated bull spermatozoa. Furthermore, the co-supplementation of GSH with SeNPs effectively protected bull spermatozoa from cryoinjury as expressed by promoting semen motility, viability, mitochondrial activity, plasma membrane integrity, and acrosome integrity. Finally, the enhanced antioxidant capacity and embryonic development potential in the frozen-thawed bull spermatozoa cryopreserved by co-supplementation of GSH with SeNPs further confirmed the synergistic protective effect of co-supplementation of GSH with SeNPs on the cryopreservation of bull semen.

Whole-genome sequencing of Histophilus somni strains isolated in Russia.

Histophilus somni is a Gram-negative bacterium belonging to the Pasteurellaceae family that can cause bovine histophilosis. Histophilus may act as a commensal or opportunistic bacterial cattle pathogen. Comparing genomes of the pathogenic strain 2336 with the non-pathogenic preputial 129Pt isolate revealed some putative virulence factors. The study of the complete genomes of H. somni strains circulating in Russia has never been conducted before. This study aimed to identify genetic features of the H. somni strains isolated in Russia and evaluate the possibility of using strains for vaccine development. Three strains of H. somni were isolated from different sources. Strain 188-VIEV was isolated from a vaginal swab sample of cattle with endometritis. 532-VIEV and 551-VIEV were cultured from the cryopreserved bull semen samples imported from Canada. Histophilus somni strain ATCC 700025 provided by ATCC (American Type Culture Collection) was also used in the study. DNA extraction was performed using QIAamp DNA Mini Kit (QIAGEN, USA). The whole-genome sequencing of the four strains was performed using Illumina Miseq. The comparison of the resulting sequences with the complete genomes of H. somni 2336 and 129Pt, and detection of the resistance genes and virulence factors, was performed using the ResFinder and Virulence Factor Database web services. The genome size of the samples varied from 1.9 to 2.3 Mb. The number of coding sequences varied from 1795 to 2256. The average sequence density was 90%. The total guanine-cytosine (GC) content was 36.8%-37.2%, which coincided with data previously obtained for H. somni. Three out of four studied strains encoded putative virulence factors such as filamentous hemagglutinin homologs, lipooligosaccharide biosynthesis proteins, and proteins involved in iron transport and utilization. The Ser83Ile substitution was identified in the DNA topoisomerase II (gyrA) in H. somni strains 532-VIEV and 551-VIEV cultured from bull semen which led to resistance to fluoroquinolones. The gene (AAC-6-Ia + APH-2'') encoding a bifunctional aminoglycoside modification enzyme was detected in strain 551-VIEV. Strains with virulence genes identified could be candidates for designing vaccines and potentially represent antigen sources. The results show that antibiotic-resistant H. somni can be spread with semen used for artificial insemination.

Evaluation of the in vitro cryopreservative performance of Juniper berry oil (Juniperus communis) on frozen-thawed bull semen

This study aimed to reveal the effects of juniper (Juniperus communis) berry oil (JB) when added to the Tris extender, on bull (Holstein) sperm motility characteristics, abnormal sperm ratio, DNA fragmentation and some oxidative stress markers. The pooled ejaculate was divided into five equal parts as control (C) and Tris diluent containing 25, 50, 100, 200 μg/ml JB. Diluted semen was incubated at 4 °C for 4 h and then frozen in liquid nitrogen (−196 °C). After freezing-thawing, motility characteristics were determined using a sperm analyzer system. The results showed that the 25 μg/ml dose of JB had an improving effect on overall sperm motility (P &lt; 0.05). The total abnormal spermatozoa ratio was not significantly different between the groups (P &gt; 0.05). The JB25 and JB50 groups had higher DNA integrity than the other groups (P &lt; 0.05). This finding was supported by low malondialdehyde levels (P &lt; 0.05). Especially the 25 μg/ml dose of JB was found to have a remedial effect on DNA integrity, as well as increasing progressive and total motility rates. This study has revealed new information on the effects of JB on bull semen cryopreservation. Juniper berry oil may be used as an antioxidant in bull semen freezing.

Silver nanoparticle as an alternate to antibiotics in cattle semen during cryopreservation.

The proposed study was to determine if the silver nanoparticles can be used as potential antimicrobial agents and can replace the use of conventional antibiotics in semen without affecting the motility and fertility of semen. The silver nanoparticles prepared by chemical reduction method were confirmed by determination of the wavelength of surface plasmon resonance peak and further characterized using Zetasizer by determining their size, polydispersity index, and zeta potential. The nanoparticles were assessed for antibacterial activity and their concentration was optimized for use in semen extender for cryopreservation. Cryopreserved semen was further evaluated for seminal parameters, antioxidant parameter, and microbial load. Prepared silver NPs showed a plasmon resonance peak at 417 nm wavelength. NPs were found to possess antibacterial activity and were supplemented in semen extender @ 125 and 250 µg/ml for semen cryopreservation. There was a significant increase in pre and post-freezing motility and other seminal parameters. The microbial load of frozen-thawed semen of control and supplemented groups were well within the permissible limits. Lipid peroxidation levels were reduced in NPs supplemented groups, and reactive oxygen species (ROS) levels were significantly reduced in semen supplemented with 125 µg/ml NPs. Thus it can be conclude that silver NPs can be successfully used as a substitute for antibiotics in cattle bull semen cryopreservation with good antimicrobial activity and no adverse effects on sperm characteristics.

Machine learning and hypothesis driven optimization of bull semen cryopreservation media

Cryopreservation provides a critical tool for dairy herd genetics management. Due to widely varying inter- and within-bull post thaw fertility, recent research on cryoprotectant extender medium has not dramatically improved suboptimal post-thaw recovery in industry. This progress is stymied by the interactions between samples and the many components of extender media and is often compounded by industry irrelevant sample sizes. To address these challenges, here we demonstrate blank-slate optimization of bull sperm cryopreservation media by supervised machine learning. We considered two supervised learning models: artificial neural networks and Gaussian process regression (GPR). Eleven media components and initial concentrations were identified from publications in bull semen cryopreservation, and an initial 200 extender-post-thaw motility pairs were used to train and 32 extender-post-thaw motility pairs to test the machine learning algorithms. The median post-thaw motility after coupling differential evolution with GPR the increased from 52.6 ± 6.9% to 68.3 ± 6.0% at generations 7 and 17 respectively, with several media performing dramatically better than control media counterparts. This is the first study in which machine learning was used to determine the best combination of constituents to optimize bull sperm cryopreservation media, and provides a template for optimization in other cell types.

Evaluation of Cryoprotective, Cryocapacitation Inhibitory and Fertility Enhancing Potential of RU-486, Sericin and Taurine in Cattle and Buffalo Semen

Background: Artificial insemination with frozen semen is of great value for livestock breeding and improvement. However, the procedure of cryopreservation leads to varying degree of oxidative and cryodamage to sperms deteriorating its quality, capacitation status and fertility. Hence, a variety of protocols, cryoprotectants and additives have been tried to protect sperms from such damages. This study was aimed to evaluate the antioxidant and cryocapacitation inhibitory potential of Mifepristone, Sericin and Taurine in routine extender on cryopreserved bull semen including in vivo fertility. Methods: Semen ejaculates of 3 Gir and 3 Murrah bulls with greater than 75% initial motility were split-diluted @ 100 million spermatozoa ml-1 using TFYG extender without and with Mifepristone (10 µg/ml), Sericin (5 mg/ml) and Taurine (4 mg/ml) and frozen in LN2 using a programmable biofreezer. The freshly diluted as well as frozen-thawed samples were assessed for sperm motility, viability, plasma membrane integrity, CTC (chlortetracycline) fluorescence assay and seminal plasma oxidative markers. The straws frozen as above were used to inseminate 100-375 cows and/or buffaloes with each treatment involving well trained field AI technicians. Conception rates were determined based on confirmation of pregnancy per-rectum 45-60 days after first AI in non-return cases. Result: The mean percentages, on dilution and at post-thaw stage in particular, of motile, live, HOS reactive and non-capacitated sperm were significantly (p less than 0.01) higher with reduced lipid peroxidation (MDA, SOD and GPx) whereas capacitated and acrosome reacted sperm were lower in Mifepristone supplemented extender than in control extender and the values for Sericin and Taurine fortified extender were intermediary in both the species. The first AI conception rates obtained for Gir and Murrah bull semen cryopreserved in control extender and extender supplemented with Mifepristone, Sericin and Taurine were 44.48, 51.11, 47.22 and 48.72% in cattle and 44.44, 55.40, 51.57 and 52.05% in buffaloes, respectively, suggesting that all additives used and Mefipristone in particular can be advantageously incorporated in the semen extender for cryopreservation of both cattle and buffalo semen.

Glycine Improved Cryopreserved Spermatozoa Quality in Achai Bull.

Achai is a small size cattle breed, resilient to harsh and cold environment. Cryopreservation of Achai bull semen may help to improve its genetics and preserve the germplasm. Reactive oxygen species (ROS) affects the structural and functional integrity of the spermatozoa. During freezing and thawing processes, the ROS make changes in the spermatozoa quality parameters and reduce total antioxidant capacity (T-AOC) of semen that is considered as marker of oxidative stress. This study was designed to determine the effect of glycine along with vitamin E on post-thawed spermatozoa quality and total antioxidant capacity in Achai cattle. The semen collection was done twice a week from four mature fertile Achai cattle bulls (n = 4). The glycine was utilized as 0 mM, 5 mM, 10 mM, 15 mM, and 20 mM along with vitamin E @ 2.3 mM added constantly in each concentration. The control group contained all extenders except glycine. The results revealed that post-thawed spermatozoa motility was found significantly higher (P < 0.05) at 10 mM as compared to 5 mM, 15 mM, and 20 mM. Compared with control group, glycine concentration at 10 mM and other concentrations increased progressive and fast motility (%), curvilinear, straight line, and average path velocity (μm/s). Moreover, beat cross frequency (Hz) was higher (P < 0.05), and post-thaw viability (%), plasma membrane integrity, and mitochondrial membrane potential were significantly higher (P < 0.05) at 10 mM of glycine concentration in comparison to control and other glycine concentrations. Besides, acrosome integrity (%) and DNA integrity (%) as well as post-thawed T-AOC were also significantly higher (P < 0.05) at 10 mM of glycine concentration as compared to other glycine concentrations and control group. It is concluded that 10 mM of glycine along with vitamin E @ 2.3 mM improved cryopreserved semen quality of Achai bull.

Sodium dodecyl sulphate, N-octyl β-D glucopyranoside and 4-methoxy phenyl β-D glucopyranoside effect on post-thaw sperm motion and viability traits of Murrah buffalo (Bubalus bubalis) bulls

Sodium Dodecyl Sulphate (SDS), N-Octyl β-D Glucopyranoside (NOG), 4-Methoxy Phenyl β-D Glucopyranoside (4-MPG) as ice recrystallization inhibitors were added to Tris Egg Yolk Glycerol (TEYG) semen extender for cryopreservation of semen of buffalo bulls. Post-thaw sperm motion and viability traits were evaluated. Pilot study involved six semen ejaculates (2 ejaculates/bull, from three bulls); second experiment was conducted using twenty seven semen ejaculates (9 ejaculates/bull, from 3 bulls) and in third experiment three semen ejaculates (one bull) were used. Eight concentrations of SDS (2, 1, 0.5, 0.25, 0.15, 0.125, 0.0625 and 0.0312%), twelve concentrations of NOG (33, 22, 11, 5.5, 2.5, 0.75, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 mM), and, eleven concentrations of 4-MPG (220, 165, 110, 55, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mM) were supplemented in TEYG semen extender to evaluate the post-thaw sperm motility and viability traits. Computer Assisted Sperm Analysis (CASA) was used to measure the kinetic and functional parameters for sperm motion traits, Hypo Osmotic Swelling Test (HOST) for sperm plasma membrane integrity, Eosin Nigrosin staining for viability and Rose Bengal staining for sperm abnormalities for all the experiments except for pilot study where only Total Motility (TM) and Rapid Progressive Motility (RP) were evaluated. Three freezing protocols; i) Normal P24 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −40 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C); ii) Moderate P25 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −50 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C); and iii) Rapid P26 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −60 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C) were evaluated using SDS 0.125% in TEYG semen extender. SDS ≤0.125%, NOG ≤0.0625 mM and 4-MPG ≤ 3.125 mM in TEYG buffalo semen extender improved significantly (p < .05) the kinetic and functional parameters as compared to the other Ice Recrystallization Inhibitors (IRIs) concentrations used for cryopreservation of buffalo bull semen in the pilot study. SDS 0.125% supplementation was the best IRI among all which resulted in improved kinetic and functional parameters of bull semen in second experiment. Conclusion was drawn that buffalo bull semen cryopreservation using sodium dodecyl sulphate, 0.125% as IRI in TEYG semen extender along with freezing protocol P 25 revealed optimum kinetic and functional parameters for post-thaw spermatozoa.

In Vitro Supplementation of Glycine Improves Quality of Cryopreserved Bull Semen

Process of cryopreservation induces cell damages due to thermal and osmotic shocks. Addition of amino acids as cryoprotective agents in extender can reduce these damages. Therefore, present study was designed to evaluate the effect of glycine supplementation in extender on freezability of cryopreserved bull semen. For this purpose, three adult Sahiwal bulls were selected for semen collection by artificial vagina having 42 C (three weeks) (replicates; n=18). Collected semen was quickly shifted to laboratory for initial evaluation. Qualified semen ejaculates (&gt;60% motility, &gt;1mL volume, &gt;0.5billion/mL conc.) were diluted in tris extender having 0 (control), 5, 10, 15 and 20mM of glycine. Semen was cooled to 4 C in 2 hours and kept for 4 hours for equilibration. Semen was filled in French straws at 4 C and kept in liquid nitrogen container. Highest percentages of post thaw sperm progressive motility and acrosome integrity were observed in extender containing 5mM of glycine compared to other treatments and control (P&lt;0.05). However, sperm viability, chromatin integrity and plasma membrane integrity remained same. In conclusion, glycine addition at 5mM in extender improves quality of cryopreserved bull semen. Keywords: Glycine, amino acid, cryopreservation, bull semen.

Fertility comparison of frozen bull semen stored in cryogenic deep freezer (−152 °C) and LN2 container

The present study aimed to use cryogenic deep freezers that could be a feasible alternative for cryopreserved semen storage. A total of 284 straws from three Simmental bulls and 272 Simmental cows were used. The experimental group consisted of 151 semen straws that were stored at −152 °C for a week. Moreover, the control group consisted of 133 semen straws that were stored at −196 °C. Firstly, two samples per bull (n = 6) were examined in terms of sperm kinetic parameters by CASA. Furthermore, plasma membrane, acrosome integrity (PMAI) and high mitochondrial membrane potential (HMMP) were analyzed by flow cytometry. Then, artificial inseminations were performed on Simmental cows with 272 straws belonging to two groups. Then, 56th-day Non-return Rate (NRR56) was determined. All spermatological data were subjected to a linear mixed model. Chi-Square test was performed to NRR56 between storage temperature groups. Also, logistic regression analysis was used to examine the effect of bull, storage temperature and age of cows on pregnancy status. While age of cows was included in the final logistic regression model, effect of bull x storage temperature was not included because it was found as non-significant. The post-thaw PMOT and STR of cryopreserved bull semen, which was stored at −152 °C, had lower and statistically significant values (p < 0.05). However, frozen bull semen, which were stored at −152 °C, kept its fertility ability as which stored at −196 °C. Besides, NRR56 of semen stored at −152 °C and −196 °C were detected as 57.24% (83/145) and 55.91% (71/127), respectively (p > 0.05). Nevertheless, these results should be supplemented with more pre-freezing and post-thaw sperm quality analyses and more fertility data for increasing the accuracy of the method.

Oligomeric Proanthocyanidins and Bamboo Leaf Flavonoids Improve the Quality of Bull Semen Cryopreservation.

It is important to inhibit oxidative stress to maintain sperm motility during cryopreservation. The present study was performed to investigate the effects of supplementing oligomeric proanthocyanidins (OPC) and bamboo leaf flavonoids (BLF) or their combination as an extender for Simmental bull semen freezing. OPC, BLF, or their combination were added to the frozen diluent of bovine semen. Afterwards, computer-assisted semen analysis (CASA), detection of membrane functionality, acrosome integrity, mitochondrial integrity, CAT, SOD, GSH-PX, MDA, and ROS were conducted. The results showed that adding 50 mg/L OPC or 4 mg/L BLF could improve the quality of frozen sperm. Compared with 50 mg/L OPC alone, the combination of 50mg/L OPC and 2 mg/L BLF significantly increased the kinematic parameters of sperm, and sperm CAT, GSH-PX and SOD levels (p < 0.05), whereas the MDA of sperm was decreased (p < 0.05). These results indicated that compared to the addition of 50 mg/L OPC alone, a combination of 50 mg/L OPC and 2 mg/L BLF could further improve the quality of frozen semen. The results could provide theoretical data support for the development of a new protective agent and are significant for the cryopreservation of bovine semen in the future.

Cryopreservation of Nili-Ravi buffalo bull semen: yearly postthaw analyses to depict climatic effects

The present study was aimed to observe the postthaw quality of Nili-Ravi buffalo bull semen cryopreserved during heat stress (hot summer months) and normal climatic conditions (fall, winter, and spring months). For this purpose, four (n = 4) adult regular donor Nili-Ravi buffalo bulls were selected to collect and cryopreserve their semen throughout the period of an entire year. The cryopreservation of semen was done following standard freezing protocol. The semen doses cryopreserved during heat stress and normal climatic condition were thawed to determine the sperm motility, motion kinetics, viability, plasma membrane integrity, acrosomal integrity, and morphological abnormalities (head, mid-piece, and tail). The results showed that sperm motility and sperm motion kinetics did not alter (p > 0.05) across heat stress and normal climatic period. The sperm head, mid-piece and tail abnormalities did not differ (p > 0.05); however, total sperm morphologic abnormalities were higher (p < 0.05) during heat stress period, whereas the lower (p < 0.05) sperm viability, sperm plasma membrane, and acrosomal integrity were observed during heat stress period. In conclusion, the total morphologic abnormalities, viability, and acrosomal and plasma membrane integrity of buffalo bull sperm cryopreserved during hot summer months are compromised due to heat stress.

Combined Effect of Flaxseed Oil and Vitamin E Supplementation During Bull Semen Cryopreservation on Sperm Characteristics.

The present study was performed to investigate the effects of supplementing flaxseed oil (FO) or vitamin E (VE) or their combination to an extender for Simmental bull semen cryopreservation. In experiment 1, different concentrations of FO (0, 10, 100, and 1000 ng/mL) and VE (0.05, 0.1, and 0.2 mg/mL) were added to the extenders. In experiment 2, FO, VE, and FO + VE were added and a control group was included. Sperm viability, motility, motion parameters, acrosome integrity and membrane integrity, endogenous antioxidant indices, reactive oxygen species, and malondialdehyde levels were evaluated after semen thawing. A higher percentage of viability, motion parameters, endogenous antioxidant indices, and membrane integrity was observed after supplementation with 10 ng/mL FO or 0.1 mg/mL VE compared with the control group (p < 0.05). Also, combined supplementation of 10 ng/mL FO +0.1 mg/mL VE further improved the quality of frozen-thawed sperm by regulating viability, motion parameters, membrane integrity, and endogenous antioxidant indices compared with the FO or VE alone (p < 0.05). These results indicated that FO (10 ng/mL) + VE (0.1 mg/mL) could further improve the protective effects on bull sperm post-thaw.