Post-thaw Semen Samples Research Articles

Investigation of the efficacy of different cryoprotectants in the freezing of testicular tissue and epididymal sperm: Spermatological parameters, tissue viability and PARP-1 gene expression

The presented study covers testicular tissue and epididymal spermatozoa cryopreservation processes in bulls and aims to investigate the effects of these applications on spermatological parameters, cell viability in testicular tissue, and the expression of the PARP-1 gene, a DNA repair enzyme. Testes of 20 bulls over 2 years old, slaughtered in a slaughterhouse, were used in the study. After spermatological evaluations, the semen obtained from the cauda epididymis was frozen in liquid nitrogen vapor according to the straw method and stored in liquid nitrogen (−196 °C). Testicular tissue pieces obtained from the testicles were frozen by the slow freezing method in cryotubes in diluents containing Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) cryoprotectants and stored in liquid nitrogen (−196 °C). The total motility (TM) (85.89 ± 12.83 %), progressive motility (PM) (54.02 ± 15.77 %), and kinematic parameter values of fresh sperm were significantly higher compared to the TM (57.62 ± 13.13 %), PM (29.60 ± 10.76 %), and kinematic parameter values after thawing (P < 0.05). Significant decreases in plasma membrane integrity (PMI) and viability and an increase in chromatin condensation and morphological disorders in the head, middle part, and tail regions were observed in post-thaw semen samples (P < 0.05). When the effects of DMSO and EG on cell viability after thaw in frozen testicular tissue were evaluated, it was observed that the cell viability values of testicular tissues frozen with EG (45.70 ± 10.00) were statistically significantly lower than those frozen with DMSO (51.20 ± 7.70) (P < 0.05). When the effects of both cryoprotectants on gene expression in tissue and semen samples were examined, it was determined that gene expression increased on average 0.19 ± 0.27 times in the tissue samples in the DMSO group compared to fresh tissue samples and 0.17 ± 0.19 times in the tissue samples in the EG group. It was determined that gene expression levels increased by an average of 1.20 ± 1.08 times in post-thaw epididymal spermatozoa samples compared to fresh semen samples. The results show that cryopreservation can activate cellular repair mechanisms by stimulating PARP-1 gene expression and affect gene expression by activating specific pathways in tissues and cells.

Identification of heat shock protein70-2 and protamine-1 mRNA, proteins, and analyses of their association with fertility using frozen-thawed sperm in Madura bulls.

This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers. The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) (79.04%; n = 4), and low fertility (LF) (65.84%; n = 4). mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate. Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality. HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.

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.

Sperm osteopontin mRNA expression levels and its correlation on semen quality and fertility in Madura bulls

Abstract. Rosyada ZNA, Yoelinda VT, Kaiin EM, Gunawan M, Ulum MF, Tumbelaka LITA, Solihin DD, Agil M, Gunawan A, Purwantara B. 2023. Sperm osteopontin mRNA expression levels and its correlation on semen quality and fertility in Madura bulls. Biodiversitas 24: 563-570. Osteopontin (OPN) gene transcripts influence spermatogenesis and germ cell development. Therefore, transcriptome analysis is needed to identify the fertility factor OPN in Madura bull sperm. Madura cattle are crosses originating from Bos indicus (zebu) and Bos javanicus (banteng). This study aims to examine sperm osteopontin (OPN) mRNA expression levels and its correlation with semen quality and fertility in Madura bulls. Frozen semen samples from Madura bulls were categorized as high-fertile (n: 4, average field conception rate: 78.28±3.25%) or sub-fertile (n: 4, average field conception rate: 66.73±5.01%). In post-thaw semen samples, sperm motility, viability, membrane, acrosome, and DNA fragmentation index were evaluated. OPN expression in sperm total RNA was analyzed using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). The data analysis between the two fertility groups was assessed with Student's t-test and Pearson Square. Receiver-operating characteristic analysis and diagnostic sensitivity and specificity calculations were performed. Sperm motility, acrosome intact, and DFI differ (p&lt;0.05) between groups, whereas viability and membrane plasma intact have no significance (p&gt;0.05). In high-fertile Madura bulls, OPN mRNA was upregulated (p&lt;0.05). The OPN mRNA expression had a strong correlation with the field conception rate (R: 0.807, P&lt;0.05), sperm motility, and intact acrosome (R: 0.899, p&lt;0.01; R: 0.804, p&lt;0.05) respectively. In contrast, OPN negatively correlated with sperm DFI (R: -0.764, p&lt;0.05). The OPN predicted bull fertility with 81.3% accuracy, 75% sensitivity, and 75% specificity. Thus, OPN may serve as a potential biomarker of Madura bull fertility.

Addition of autologous platelet rich plasma to semen extender enhances cryotolerance and fertilizing capacity of buffalo bull spermatozoa

Platelet rich plasma (PRP) is extensively used in regenerative medicine. Present work was aimed to investigate the effect of autologous PRP supplementation in semen freezing extender on sperm quality, antioxidant capacity, lipid peroxidation and in vitro fertilizing capacity of frozen-thawed buffalo semen and subsequent embryo developmental competence. Buffalo bulls, n = 8, were used as semen donors. Semen ejaculates were separately divided into four equal parts and extended with autologous PRP 0 (control), 2, 5 and 10% supplemented Tris-based semen extender. Extended semen samples were then cooled to 5 °C for 2h and processed for cryofreezing in French straws. Post-thawed semen samples (37 °C for 30 s) were evaluated for progressive motility (PM), structural membrane integrity (SMI), functional membrane integrity (FMI), total abnormalities (TA), and acrosome integrity (AI). Supernatant from the thawed samples was examined colormetrically for superoxide dismutase activity (SOD), total antioxidant capacity (TAC) and lipid peroxidation profile (MDA). Fertilizing capacity of post-thaw spermatozoa cryofrozen in 5% PRP extender was tested upon fertilization the buffalo oocytes in vitro. Higher (P < 0.05) post-thaw sperm quality (PM, SMI, FMI, AI) in 5% PRP semen extender, whereas TA was lower in control, 2% and 10% concentrations. Five percent PRP supplemented semen extender resulted in greater (P < 0.05) TAC and SOD, and lesser MDA levels compared to other groups. Inseminated buffalo oocytes with sperm cryofrozen in 5% PRP revealed higher fertilization, cleavage and blastocyst rate and lower polyspermy as compared to control. In conclusion, buffalo spermatozoa cryofrozen in autologous PRP supplemented semen extender enhanced cryotolerance and fertilizing potential.

Growth performance, reproductive parameters and fertility measures in young Nellore bulls with divergent feed efficiency.

The growth, sexual maturity and fertility-related parameters related of young Nellore bulls with divergent residual feed intake (RFI) raised on pasture were evaluated. After classification of 48 young males as low and high RFI (more and less efficient, respectively), the animals were evaluated for growth and reproductive parameters at 28-day intervals from 14.3 to 24.6 months of age. The semen was cryopreserved in the last sampling and fresh and post-thaw semen samples were evaluated. Low RFI bulls exhibited higher initial and final body weight (P < 0.05), but feed intake, body condition score and growth measures evaluated by carcass ultrasound were unaffected by RFI (P > 0.05). The scrotal circumference, sperm concentration, defects, and quality of fresh semen, and ultrasonographic testicular characteristics were unaffected by RFI (P > 0.05). However, velocity parameters such as average path and curvilinear velocities determined by computer-assisted sperm analysis of thawed semen submitted to the rapid thermoresistance test were improved (P < 0.05) in low RFI bulls, but this improvement in quality did not enhance in vitro sperm fertilizing ability. Our results demonstrated significant differences in metabolism and growth performance between bulls of divergent RFI. In addition, there was slight improvement in the semen quality of bulls with low RFI bulls, but this did not enhance in vitro fertilizing ability. Selection of beef bulls for RFI can be performed, which will result in economic benefits by improving the growth performance of the animals without affecting reproductive parameters.

A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs

In order to accurately analyze the possible side effects of sperm cryopreservation, an in-depth screening of post-thaw sperm status is necessary. Thus, this study aimed to identify thorough effects of sperm cryopreservation, by evaluating the integrity of all specific structures of the canine spermatozoa. Thirteen (n = 13) mature dogs of different breeds were selected. Six dogs (n = 6) were subjected to sperm cryopreservation, whereas seven dogs (n = 7) were used as semen donors to validate a simultaneous assessment of sperm plasmatic, acrosomal, and mitochondrial membranes (triple stain) by fluorescent probes. Fresh and post-thaw semen samples were evaluated through a computer-assisted analysis of sperm motility, sperm morpho-functional evaluation, triple stain and sperm DNA integrity. Post-thaw semen samples had lower total and progressive motility, as well as higher percentage of minor and major defects. Moreover, post-thaw samples had higher percentage of sperm with plasma membrane and mitochondrial damage but intact acrosome, and also sperm with simultaneous damaged plasma, acrosomal and mitochondrial membranes. Furthermore, post-thaw sperm had higher protamination deficiency and DNA fragmentation. In conclusion, cryopreservation has a broad impact in sperm morphology and function, altering motility patterns, plasma, acrosome and mitochondrial membranes integrity, as well as sperm DNA.

Kualitas post-thawing semen domba Merino dalam bahan pengencer berbasis susu skim-kuning telur yang ditambah isolat crude protein Tirosine Kinase

This study was conducted to determine the effect of crude protein tyrosine kinase (PTK) isolates supplementation into skim milk-egg yolk based diluent to maintain the quality of Merino ram spermatozoa. Four ejaculates of two Merino rams were divided into two groups for the control group (P0): Merino ram semen was diluted in skimmed milk-egg yolk based diluent, and the treatment group (P1): Merino ram semen was diluted in skim milk-egg yolk based diluent contained crude PTK 1,597 mg/ml diluent. All diluted semen was equilibrated for 2 hours at 5 °C and filled into 0.25 mL French straws. The filled straws were placed on steel racks (Cooltop, Minitube) held in liquid nitrogen vapour for 10 minutes at –140 °C, immersed immediately in liquid nitrogen at –196 °C, and stored for 48 hours for later assessment. Post-thawed semen samples were evaluated for spermatozoa motility, viability, and morphological abnormality. The results showed that the spermatozoa motility of fresh semen of Merino ram was 82.5 ± 2.89, which was qualified for freezing. The post-thawing spermatozoa motility, viability, and morphological abnormalities of Merino ram in the P1 group were 34.11 ± 3.26%, 38.00 ± 3.00%, and 12.89 ± 4.54%, respectively. It were higher (p &lt;0.05) than the control group of 24.44 ± 2.9%, 26.67 ± 3.32%, and 21.11 ± 3.02%. It was concluded that the addition of crude PTK isolates of 1.597 mg/ml skim milk-egg yolk diluent improved the quality of post-thawed spermatozoa of Merino ram.

L-Proline: An Effective Agent for Frozen and Post-thawed Donkey Semen Storage

The aim of this study was to evaluate the effects of L-proline on the extender quality of frozen and post-thawed jackass semen. Jackass (n = 6) semen samples were collected and cryopreserved in gradient concentrations (0–80 mM) of L-proline in extenders; post-thawed semen samples were cultured in L-proline medium for 10 hours at 37°C. For cryopreservation experiment I, the motile parameters, mitochondrial membrane potential (MMP), and plasma membrane, acrosome, and chromatin structure integrities of post-thawed semen were assessed. For culture experiment II, additional ROS contents were analyzed after incubation. For the fertility trial, jennies (n = 135) were divided into group I (30 mM L-proline in cryopreservation extender), group II (40 mM L-proline in culture medium), and the control. Pregnancy was diagnosed using an ultrasound scanner 30 days after ovulation. The results of experiment I showed that, motile parameters and acrosome and chromatin structure integrities of groups I and 40 mM were significantly higher than the control (P < .05). MMP of group I was significantly higher than the control and 40 mM groups (P < .05). In experiment II, after 4 hours of incubation, motile parameters, MMP, and DNA integrity in group II were significantly higher than the control (P < .05). Additionally, 40 and 80 mM L-proline in culture medium significantly reduced ROS accumulation after 4 and 10 hours of incubation (P < .05). Pregnancy rates of the control and groups I and II were 28.85%, 40%, and 36.84%, respectively. In conclusion, the extenders containing 30 to 40 mM L-proline improved both qualities of frozen and post-thawed semen, and it will be a beneficial agent for donkey frozen spermatozoa or post-thawed semen storage.

Effect of Simmental bull seminal plasma protein in egg yolk-citrate extender on Kacang buck semen fertility

The genetic resources of Indonesia's indigenous Kacang goat require preservation. Artificial insemination is expected to accelerate population increases and preserve genetic resources simultaneously. The present study was the maiden attempt for cryopreservation of Kacang buck sperm. The objectives of this study were to determine whether the supplementation of superior Simmental bull seminal plasma protein in egg yolk-citrate extender could improve the quality of post-thawed Kacang buck sperm, increase conceptions rates, and improve kidding rates. Buck semen was diluted without supplementation (T0) and with supplementation of 2.5 mg (T1) and 5 mg (T2) of Simmental bull seminal plasma protein per mL egg yolk-citrate extender. Extended semen was packed in 0.25 mL straw as cryopreserved frozen semen. Post-thawed semen samples were evaluated for viability, motility, intact plasma membranes, malondialdehyde level, and DNA fragmentation. Estrus was synchronized for sixty Kacang does, which were divided randomly into three groups and inseminated using post-thawed semen. The progesterone serum concentration of the does was measured 7 and 22 days post-insemination to detect ovulation and conception. Pregnancy was confirmed using abdominal palpation at 43 days post-insemination and by observing birth. The T1 group showed the highest (P < 0.05) post-thawed viability, motility, and intact plasma membrane. Conception, pregnancy and kidding rates were also higher in T1 than other treatment groups. In conclusion, the 2.5 mg Simmental bull seminal plasma protein supplementation per mL egg yolk-citrate extender provided the best seminal quality and fertility of post-thawed Kacang buck semen.

Effects of antioxidants on motility and DNA integrity in frozen-thawed sperm

Aims: This study aims to investigate whether the addition of the antioxidant complex including taurine, ascorbic acid, and glutathione into the cryopreservation medium affects the damage to sperm during the freezing process. Methods: Ejaculate samples of patients who applied for semen analysis to the Assisted Reproduction Unit of Private Adatip Hospital were used. Fresh samples were analyzed for standard semen quality parameters according to the World Health Organization guidelines. Samples within the normal range were evaluated for sperm DNA fragmentation using the Halosperm technique. Remaining ejaculates were washed with the gradient method before cryopreservation. Sperm samples of each patient were divided equally for freezing in a cryopreservation medium with or without the antioxidant supplementation. One month later, the samples were thawed. Post-thaw total motility and DNA fragmentation were determined for each sample. Results: Semen samples of 40 patients were analyzed. We observed decreased total motility (34.8 ±5.32 % vs. 65.5 ±6.42 %, P= 0.002) and increased sperm DNA fragmentation (52.3±5.42 % vs. 26.4±3.12 %, P= 0.002) in post-thaw semen samples following cryopreservation in comparison to fresh samples. The addition of antioxidants to the freezing medium did not have a statistically significant effect on sperm motility (38.3± 6.22 % vs. 34.8 ±5.32 %, P =0.07) and DNA damage (47.5±4.7 % vs. 52.3±5.42, P =0.08) when compared to control samples following the freezing process. Conclusions: We observed increased sperm DNA fragmentation and decreased total motility following cryopreservation. No significant improvement in sperm motility or DNA integrity was obtained after the addition of 5 µM of the antioxidants taurine, ascorbic acid, and glutathione to the freezing media.

Sexual maturity and fertility-related measures in young Nellore bulls receiving long-term dietary supplementation with rumen-protected polyunsaturated fatty acids

The objective of this study was to evaluate the effects of long-term supplementation with rumen-protected fatty acids (FA) on growth and reproductive parameters of young Nellore bulls in a grazing regime. Forty-eight young bulls were distributed into two groups: FA (supplemented with rumen-protected polyunsaturated FA); and control (control fat-free supplement). The animals were supplemented from 14.3 to 24.6 months of age and growth and reproductive parameters were evaluated at 28-day intervals. The semen was cryopreserved in the last collection and fresh and post-thaw semen samples were evaluated. Feeding FA did not affect (P > 0.05) growth, reproductive parameters (scrotal circumference, sperm concentration per mL of ejaculate, percentage of sperm defects, sperm quality and fertility in vitro), or testicular ultrasonographic characteristics. However, thawed semen from bulls fed FA exhibited better quality (P < 0.05) than control semen for the following parameters evaluated by computer-assisted sperm analysis: average path velocity [μm/s: 90.48 vs. 79.66 post-thaw and 74.81 vs. 72.80 post-rapid thermoresistance test (TRT)], straight-line velocity (μm/s: 72.37 vs. 65.20 post-thaw and 64.96 vs. 63.25 post-TRT), and curvilinear velocity (μm/s: 148.44 vs. 131.31 post-thaw and 115.68 vs. 113.35 post-TRT). In addition, feeding FA increased peripheral concentrations of testosterone, leptin, total cholesterol and high-density lipoprotein. In conclusion, the increase in testosterone concentrations in bulls fed FA was not related to variations in growth parameters and sexual maturity. In addition, post-thawing sperm velocities were enhanced by diet, however, such increases were not related to better in vitro embryo production rates.

Flaxseed oil modulates semen production and its quality profiles, freezability, testicular biometrics and endocrinological profiles in mithun

Mithun (Bos frontalis) is a unique domestic free range bovine species of North Eastern Hilly (NEH) regions of India. Effect of feed supplementation of Flaxseed oil (FSO) on semen production and its quality profiles, freezability, oxidative stress, apoptotic sperm percentage and subsequently on endocrinological profiles & scrotal and testicular biometrics in different seasons was studied in mithun. The experimental animals were divided into two groups, Gr I: Control (n = 3) and Gr II: Treatment (n = 3; Flaxseed oil @ 150 mL/day). FSO was supplemented through oral drench in the morning hours just before concentrate feeding. A total of 80 semen samples (n = 80; 20 semen samples from each season; each 10 semen samples from control and treatment groups per season) were collected, not more than twice per week in winter, spring, autumn and summer seasons. Semen quality profiles (SQPs) such as volume, sperm concentration, motility (forward progressive and total), motility & velocity profiles by computer assisted sperm analyser (CASA), viability, total sperm abnormality, acrosome integrity, plasma membrane & nuclear abnormality and apoptotic sperm percentage were estimated in fresh semen. Along with SQPs measured in fresh semen, motility in estrus bovine cervical mucus (bovine cervical mucus penetration test; BCMPT) and mitochondrial membrane potential (MMP) by JC-1 stain were determined in the post-thawed semen samples. Biochemical profiles (aspartate aminotransferase; AST, alanine aminotransferase; ALT, total cholesterol; CHO), antioxidant profiles (superoxide dismutase; SOD, catalase; CAT, glutathione; GSH, total antioxidant capacity; TAC) and oxidative stress profile (malondialdehyde; MDA) were estimated in fresh semen whereas AST, ALT, lactate dehydrogenase (LDH), TAC and MDA were estimated in the frozen thawed semen samples. Endocrinological profiles such as follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, cortisol and thyroxin and scrotal circumference (SC) & testicular biometrics were measured in both groups in different seasons. Result revealed a significant (p < 0.05) improvement in motility (total & forward progressive, motility & velocity by CASA and vanguard distance in cervical mucus), viability, intactness of acrosome & plasma membrane, MMP, antioxidant profiles and reduction in total sperm and nuclear abnormalities, reduction in leakage of intracellular enzymes and reduction in oxidative stress profile and reduction of apoptotic sperm percentage were observed in FSO supplemented than in un-supplemented control group accordingly in fresh and post thawed semen samples. Blood FSH, LH, testosterone and thyroxin concentration were significantly (p < 0.05) increased and cortisol concentration was significantly (p < 0.05) decreased in FSO supplemented group than in unsupplemented control group. Similarly, SC and testicular biometrics were increased significantly (p < 0.05) in supplemented than unsupplemented group for different seasons and significantly (p < 0.05) higher in winter and spring than in summer season in the experimental groups. It can be concluded from the study that supplementation of FSO can effectively be utilized to improve the antioxidant profiles, reduction of oxidative stress with cascading beneficial effects on SQPs and fertility status of the mithun bull.

Effect of Mare Colostrum in Extenders for Freezing Stallion Semen

This study aimed to evaluate the addition of mare colostrum in stallion freezing extenders to improve sperm quality. First, colostrum samples were collected from four mares after the foal's birth and their composition was determined. Ejaculates were collected from nine fertile stallions. Sperm samples were pooled, diluted, and cryopreserved into three experimental extender groups: Lactose-based extender supplemented with mare colostrum (20%), lactose-based extender supplemented with egg yolk (20%), and BotuCrio. The quality of the post-thaw semen samples were evaluated assessing sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodine (PI) stain, acrosome integrity by fluorescein isothiocyanate and peanut agglutinine (FITC-PNA) and PI stain, plasma membrane functionality by hypo-osmotic swelling (HOS) test, and DNA denaturation by acridine orange (AO) test. There were no significant differences in the percentages of total motility, acrosome integrity, and DNA fragmentation among the extenders after thawing. Kinematics parameters showed significantly higher values in BotuCrio than in lactose extenders (P < .05). BotuCrio and lactose colostrum extender yielded significantly better rates for HOS-test, linearity, straightness, and wobble than egg-yolk extender (P < .05). However, in relation to sperm viability, lactose egg yolk extender showed significantly better results in comparison to the others seminal experimental media (P < .05). In conclusion, the incorporation of mare colostrum into cryopreservation media protected the sperm against cold-shock; therefore, it may be a good cryoprotectant agent alternative in extenders for freezing stallion semen.

Administration of slow release exogenous melatonin modulates oxidative stress profiles and in vitro fertilizing ability of the cryopreserved mithun (Bos frontalis) spermatozoa

Mithun (Bos frontalis) is a unique domestic free range bovine species of North Eastern hilly regions of India. The present study was designed to assess the seasonal effect of slow release exogenous melatonin (MT) implant on semen quality parameters (SQP) and in vitro zona binding ability (IVZ) of spermatozoa. The experimental animals were divided into Gr I: Control (n = 5) and Gr II: Treatment (n = 5; melatonin implant @ 18mg/50 kg bwt). A total of 20 semen samples/group in winter, spring, autumn and summer seasons (n = 160), twice per week were collected. Following cryopreservation, samples were evaluated for motility parameters (forward progressive, mobility & velocity by computer assisted sperm analyser (CASA), viability, acrosome integrity, plasma membrane and nuclear abnormality, functional status of mitochondria, enzymatic, antioxidant and oxidative profiles, and IVZ. The study revealed significant (p < 0.05) improvement in total motility, viability, acrosome-, plasma membrane-, and nuclear-integrity, and antioxidant profiles; with highest values in spring and lowest in summer season in the fresh semen in Gr II than the Control. A significant (p < 0.05) improvement in motility parameters, membrane potential of mitochondria, antioxidant profiles and reduction in sperm and nuclear abnormalities, leakage of intracellular enzymes and oxidative stress and IVZ index & binding percentage in post-thaw semen samples in melatonin supplemented than in un-supplemented control group was observed. It can be concluded from the study that slow-release melatonin supplementation can be effectively utilized to improve the antioxidant profiles and reduction of oxidative stress, with cascading beneficial effects on semen quality parameters and fertility status of the mithun bull.

Stallion sperm selection prior to freezing using a modified colloid swim-up procedure without centrifugation

The aims of this study were to: 1) develop a new method for stallion sperm selection using a modified swim-up procedure through a colloid and 2) evaluate its impact in good quality ejaculates from bad freezers in comparison to methods involving centrifugation such as single layer centrifugation and sperm washing. Ejaculates were processed before freezing using three different procedures: sperm washing (SW), colloid single layer centrifugation (SLC) and a modified colloid swim-up (SU). After semen processing, sperm recovery rates were measured and sperm were frozen. Post-thaw sperm motility (assessed by computer-assisted sperm analysis), normal forms and plasma membrane integrity (evaluated under bright-field and fluorescence microscopy respectively), and DNA fragmentation (assessed by the Sperm-Halomax kit) were compared between treatments. Sperm recovery rates were similar between SU and SLC but lower than SW. Sperm motility after thawing was lower in SU in comparison to SLC and SW, maybe due to the incomplete removal of seminal plasma before freezing. Sperm DNA fragmentation was lower in SU and SLC selection methods, particularly in SLC selected samples during the first 6h of incubation. The remaining sperm parameters assessed were similar among treatments. In conclusion, SLC is more suitable than SW and SU to process stallion semen prior to freezing, in particular when sperm DNA damage is suspected. Further studies are needed in order to determine the potential benefits of SU in samples where centrifugation is not necessary, such as epididymal sperm, ejaculate fractioning or post-thaw semen samples.

Cumene hydroperoxide induced changes in oxidation-reduction potential in fresh and frozen seminal ejaculates.

Oxidation-reduction potential (ORP) is a newer integrated measure of the balance between total oxidants (reactive oxygen species-ROS) and reductants (antioxidants) that reflects oxidative stress in a biological system. This study measures ORP and evaluates the effect of exogenous induction of oxidative stress by cumene hydroperoxide (CH) on ORP in fresh and frozen semen using the MiOXSYS Analyzer. Semen samples from healthy donors (n=20) were collected and evaluated for sperm parameters. All samples were then flash-frozen at -80°C. Oxidative stress was induced by CH (5 and 50μmoles/ml). Static ORP (sORP-(mV/106 sperm/ml) and capacity ORP (cORP-μC/106 sperm/ml) were measured in all samples before and after freezing. All values are reported as mean±SEM. Both 5 and 50μmoles/ml of CH resulted in a significant decline in per cent motility compared to control in pre-freeze semen samples. The increase in both pre-freeze and post-thaw semen samples for sORP was higher in the controls than with 50μmoles/ml of CH. The change from pre-freeze to post-thaw cORP was comparable. The system is a simple, sensitive and portable tool to measure the seminal ORP and its dynamic impact on sperm parameters in both fresh and frozen semen specimens.

New approach to assess sperm DNA fragmentation dynamics: Fine-tuning mathematical models

BackgroundSperm DNA fragmentation (sDF) has been proved to be an important parameter in order to predict in vitro the potential fertility of a semen sample. Colloid centrifugation could be a suitable technique to select those donkey sperm more resistant to DNA fragmentation after thawing. Previous studies have shown that to elucidate the latent damage of the DNA molecule, sDF should be assessed dynamically, where the rate of fragmentation between treatments indicates how resistant the DNA is to iatrogenic damage. The rate of fragmentation is calculated using the slope of a linear regression equation. However, it has not been studied if sDF dynamics fit this model. The objectives of this study were to evaluate the effect of different after-thawing centrifugation protocols on sperm DNA fragmentation and elucidate the most accurate mathematical model (linear regression, exponential or polynomial) for DNA fragmentation over time in frozen-thawed donkey semen.ResultsAfter submitting post-thaw semen samples to no centrifugation (UDC), sperm washing (SW) or single layer centrifugation (SLC) protocols, sDF values after 6 h of incubation were significantly lower in SLC samples than in SW or UDC. Coefficient of determination (R2) values were significantly higher for a second order polynomial model than for linear or exponential. The highest values for acceleration of fragmentation (aSDF) were obtained for SW, followed by SLC and UDC.ConclusionSLC after thawing seems to preserve longer DNA longevity in comparison to UDC and SW. Moreover, the fine-tuning of models has shown that sDF dynamics in frozen-thawed donkey semen fit a second order polynomial model, which implies that fragmentation rate is not constant and fragmentation acceleration must be taken into account to elucidate hidden damage in the DNA molecule.

Novel insights into the role of cell-free seminal mRNAs on semen quality and cryotolerance of spermatozoa in bulls (Bos taurus).

The aim of the present study was to ascertain the effectiveness of seminal plasma mRNAs as markers to assess the reproductive performance of bulls. Semen samples (33 ejaculates) from 11 bulls were evaluated for sperm kinematic and functional parameters. Total RNA was isolated from cell-free seminal (cfs) using TRIzol LS reagent and the concentration of cfs-RNA was 24.4±2.3µgmL-1 seminal plasma. The cfs-RNA was fragmented to a size of 25-500bp. Of the cfs-mRNAs screened using real time PCR, expression of protamine 1 (PRM1) was positively (P<0.05) associated with the mitochondrial membrane potential of raw semen, whereas expression of Fas Ligand (FASLG) was negatively (P<0.05) associated with sperm velocity, membrane integrity and chromatin distribution in post-thaw semen samples. The percentage of Type A spermatozoa (amplitude of lateral movement of head >2.5μm and straightness >85%) in raw semen was positively (P<0.05) associated with bone morphogenetic protein 2 (BMP2), ubiquitin conjugating enzyme E2D3 (UBE2D3), tumour-associated necrotic factor-associated death domain (TRADD) and caspase-3 (CASP3) expression. Nerve growth factor (NGF) expression was positively (P<0.05) associated with the maintenance of post-thaw functional membrane integrity in spermatozoa and could be used to assess the cryotolerance of bull semen. In conclusion, the expression of cfs mRNAs can be used to assess the reproductive performance of males and to predict the sensitivity of spermatozoa to cryoinjury.

Minimization of apoptosis-like changes in cryopreserved buffalo bull sperm by supplementing extender with Bcl-2 protein.

Aim:This study was aimed at evaluating the anti-apoptotic effects of Bcl-2 protein in cryopreserved buffalo bull sperm.Materials and Methods:A total 10 ejaculates from two buffalo bulls (5 each) were collected using artificial vagina method, and semen was evaluated using a standard protocol. Semen was extended by Tris egg yolk extender supplemented with Bcl-2 protein at 5, 10, and 15 µM. Semen was cryopreserved at ultra-low temperature using traditional vapor freezing method. Pre-freeze and post-thaw semen samples were evaluated for percent motility, viability, hypo-osmotic swelling test (HOST) reactive sperms; status of mitochondrial membrane activity and status of sperm phospholipase A1 and phospholipase A2 activity.Results:There were no significant effects of Bcl-2 protein supplementation on pre-freeze sperm quality. Percent motility and active mitochondria in post-thaw Bcl-2 supplemented and control groups were also similar. However, viable sperms were significantly (p<0.05) higher (74.29±4.23%) in Bcl-2 supplemented group (5 µM) as compared to control (51.6±5.77%). The proportion of HOST reactive sperms was also higher (63.1±6.73%) in Bcl-2 supplemented (5 µM) group as compared to control (50.7±6.98%). The sperm with low PLA activity (non-apoptotic) was significantly (p<0.05) higher in all the supplemented doses of Bcl-2 protein, i.e., at 5 µM (73.42±5.79%), 10 µM (75.51±6.22%), and 15 µM (74.78±5.89%) as compared to control (60.23±4.45%). We found that Bcl-2 protein supplementation at 5 µM dose improved the post-thaw semen quality indicated by higher viability, HOST reactive sperms, and sperm with low PLA activity (non-apoptotic sperms).Conclusion:Bcl-2 protein supplementation exerts its protective effect on spermatozoa against apoptosis-like changes developed during cryopreservation.