Post-thaw Semen Quality Research Articles

Quercetin supplemented casein-based extender improves the post-thaw quality of rooster semen

The advantageous influence of quercetin (Q) supplementation in an extender has not yet been evaluated for rooster semen cryopreservation. This research was purposely conducted in order to assess the effect of different quercetin concentrations added into an extender on the sperm quality of the rooster subsequent to a freezing-thawing process. After the freezing-thawing process, spermatozoa quality parameters (membrane functionality, acrosome integrity, motility, viability, and abnormal morphology), endogenous enzymes (SOD, CAT, and GPx), mitochondrial activity, DNA fragmentation index, lipid peroxidation (MDA), and ROS were all evaluated. A total of 75 neat pooled ejaculates (3 ejaculates/rooster) were collected from 25 arbor acres roosters (24 wks) twice a week using abdominal massage technique, then divided into five equal aliquots and diluted with an extender containing different doses of Q (CS-Q) as follows: casein extender without Q (control only), casein extender containing 0.040 mg/mL quercetin (CS-Q 0.040), 0.020 mg/mL quercetin (CS-Q 0.020), 0.010 mg/mL quercetin (CS-Q 0.010), and 0.005 mg/mL quercetin (CS-Q 0.005). Our results depicted that adding to the extender with a 0.010 mg/mL Q enhanced (P < 0.01) sperm motility, membrane function, viability, mitochondrial activity, intact acrosome (P < 0.05), SOD (P < 0.001), CAT, and GPx (P < 0.01) compared to the control group at post-thaw. Compared to the control group and other treatment groups after the freeze-thawing process, the addition of 0.005 mg/mL Q into the extender also showed higher (P < 0.05) improvement in the quality of sperm parameters and a higher (P < 0.01) SOD and CAT but did not improve mitochondrial activity and sperm viability. In addition, there was a lower degree of DNA fragmentation index, lower (P < 0.05) lipid peroxidation and ROS in frozen-thawed sperm treated with 0.010 mg/mL and 0.005 mg/mL Q than in control and the other treatment groups. In addition, 0.020 mg/mL Q supplementation into the extender also reduced DNA fragmentation and improved GPx activity compared to the control group at post-thaw. Different concentrations of Q 0.010 and 0.005 mg/mL added to the extender reduced the percentage of abnormal spermatozoa compared to the other groups. The results of this study showed for the first time that the inclusion of an extender with a suitable quercetin concentration of 0.010 mg/mL improved the post-thawed quality of rooster semen.

Effects of extender type on the quality of post-thaw giant panda (Ailuropoda melanoleuca) semen

Sperm cryopreservation is an essential approach for assisted reproduction and genetic resources conservation in captive giant pandas. Cryopreservation, however, leads to a significant decrease in sperm quality and, consequently, a low fertilization rate. Therefore, it is mandatory to disclose more suitable and efficient freezing strategies for sperm cryopreservation. In the present study, we compared for the first time the performance of two commercial freeze extender (INRA96 versus TEST) freezing methods on post-thawed semen quality. Semen cryopreserved with the INRA96 showed better total motility (73.00 ± 4.84% vs 57.56 ± 3.60%, P < 0.001), membrane integrity (60.92 ± 2.27% vs 40.53 ± 2.97%, P < 0.001) and acrosome integrity (90.39 ± 2.74% vs 84.26 ± 4.27%, P < 0.05) than stored with TEST. There was no significant difference in DNA integrity after thawing between the two extenders (95.69 ± 3.60% vs 94.26 ± 4.84%). In conclusion, the INRA96 method showed to be better for giant panda sperm cryopreservation and should therefore be recommended for use in order to increase success of artificial insemination.

Comparative transcriptome analysis digs out genes related to antifreeze between fresh and frozen–thawed rooster sperm

The objective of this study was to investigate differences in mRNA expression between fresh and frozen–thawed sperm in roosters. In trial 1, gene expression profiles were measured using microarray with Affymetrix GeneChip Chicken Genome Arrays. The results showed that 2,115 genes were differentially expressed between the 2 groups. Among these genes, 2,086 were significantly downregulated and 29 were significantly upregulated in the frozen–thawed sperm group. Gene Ontology (GO) analysis showed that more than 1,000 differentially expressed genes (DEG) of all significantly regulated genes were involved in GO terms including biological processes, molecular function, and cellular component. Kyoto Encyclopedia of Genes and Genomes analysis showed that DEG were significantly (P < 0.05) enriched on ribosome, oxidative phosphorylation, proteasome, cell cycle, oocyte meiosis, and spliceosome pathways. In trial 2, ejaculated semen was collected from 18 roosters and divided into 5 recombinant HSP90 protein–supplemented groups (0.01, 0.1, 0.5, 1, or 2 μg/mL) and one control group with no recombinant HSP90 protein supplementation to evaluate the effect of recombinant HSP90 protein in the extender on post-thaw quality of rooster semen. The results showed that post-thaw sperm viability and motility was significantly improved (P < 0.05) in the extender containing 0.5 and 1 μg/mL of recombinant HSP90 protein compared with the control. Our preliminary results will provide a valuable basis for understanding the potential molecular mechanisms of cryodamage in frozen–thawed sperm and theoretical guidance to improve the fertility of frozen–thawed chicken sperm.

Effect of dimethylformamide on sperm quality and fertilizing ability of Indian red jungle fowl (Gallus gallus murghi)

The present study investigates the efficacy of dimehtlyformamide (DMF) as a permeable cryoprotectant and its effect on quality and fertility of Indian red jungle fowl sperm. Semen was collected from eight mature roosters, pooled, divided into five aliquots and diluted with red fowl extender having DMF (0%, 4%, 6%, 8% and 10%). Diluted semen samples were cooled from 37 °C to 4 °C, 20% glycerol added to control (0% DMF), equilibrated for 10 min and filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Sperm motility, plasma membrane functionality, viability and acrosome integrity were assessed at post dilution, cooling, equilibration and freeze-thawing stage of cryopreservation. Cryopreservation stages had negative effects (P < 0.05) on semen quality parameters. Percentages of sperm motility, plasma membrane functionality, viability and acrosome integrity were recorded highest in extender having 8% DMF at post-dilution, cooling, equilibration and freeze-thawing stage. Fertility results after artificial insemination were recorded higher (P < 0.05) with 8% DMF compared to 20% glycerol. Dimehtlyformamide (8%) in red fowl extender improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol.

Gene Polymorphisms in Boar Spermatozoa and Their Associations with Post-Thaw Semen Quality.

Genetic markers have been used to assess the freezability of semen. With the advancement in molecular genetic techniques, it is possible to assess the relationships between sperm functions and gene polymorphisms. In this study, variant calling analysis of RNA-Seq datasets was used to identify single nucleotide polymorphisms (SNPs) in boar spermatozoa and to explore the associations between SNPs and post-thaw semen quality. Assessment of post-thaw sperm quality characteristics showed that 21 boars were considered as having good semen freezability (GSF), while 19 boars were classified as having poor semen freezability (PSF). Variant calling demonstrated that most of the polymorphisms (67%) detected in boar spermatozoa were at the 3’-untranslated regions (3’-UTRs). Analysis of SNP abundance in various functional gene categories showed that gene ontology (GO) terms were related to response to stress, motility, metabolism, reproduction, and embryo development. Genomic DNA was isolated from sperm samples of 40 boars. Forty SNPs were selected and genotyped, and several SNPs were significantly associated with motility and membrane integrity of frozen-thawed (FT) spermatozoa. Polymorphism in SCLT1 gene was associated with significantly higher motility and plasma membrane integrity of FT spermatozoa from boars of the GSF group compared with those of the PSF group. Likewise, polymorphisms in MAP3K20, MS4A2, and ROBO1 genes were significantly associated with reduced cryo-induced lipid peroxidation and DNA damage of FT spermatozoa from boars of the GSF group. Candidate genes with significant SNP associations, including APPL1, PLBD1, FBXO16, EML5, RAB3C, OXSR1, PRICKLE1, and MAP3K20 genes, represent potential markers for post-thaw semen quality, and they might be relevant for future improvement in the selection procedure of boars for cryopreservation. The findings of this study provide evidence indicating that polymorphisms in genes expressed in spermatozoa could be considered as factors associated with post-thaw semen quality.

Scrotal thermography to assess the seminal quality in Nelore and Girolando bulls

Bull selection by andrological examination aims to estimate the reproductive capacity of the male. Bulls of zebu origin adapt better to high temperatures than bulls of taurine origin, which may influence scrotum temperature and seminal quality due to the imbalance of testicular thermoregulation. The objective of this study was to investigate the relationship between bioclimatic variables, the temperature of body and scrotal areas, assessed with infrared thermography, and the quality of fresh and post-thawed semen in zebu Nelore bulls (Bos taurus indicus) and Girolando bulls (Bos taurus taurus x Bos taurus indicus). Bulls were kept in pickets with access to water, mineral mix and a diet supplemented with concentrate. Infrared thermographs of the scrotum, orbital globe and muzzle were performed twice a week with a Flir E40 thermal imager. For scrotal thermograms, we analyzed the temperatures of the spermatic cord, proximal and distal portion of the testes and tail of the epididymis using the Flir Tools software. Samples were collected using an artificial vagina and the ejaculates were processed and frozen in liquid nitrogen until further analyses. Data were analyzed with the Tukey test or the Kruskall-Wallis test, depending on their normal distribution. Our results showed differences (p &lt; 0.05) between the two breeds regarding the temperature in the ocular globe, spermatic cord and proximal portion of the scrotum. Nelore bulls presented lower temperature in the body and in certain regions of the scrotum compared to Girolando bulls. Seminal characteristics varied between breeds, with the Nelore breed presenting better semen. Positive correlations were observed between minor sperm defects and ventral regions of the testes and tails of the epididymis in Girolando bulls. Nelore bulls were less influenced by climatic variables and presented lower temperature in skin surface areas in the infrared thermography examination compared to Girolando bulls. Nelore bulls presented superior semen quality in both fresh and thawed samples than Girolando bulls.

Post thaw treatment of frozen buffalo semen with antioxidants vitamin C and 2-mercaptoethanol

The aim of this study was to evaluate the effect of ascorbic acid (Vitamin C) and 2-mercaptoethanol (ME) added to buffalo semen samples after thawing for 1 h and 2 h incubation. Ejaculates (n = 20) from eight Bulgarian Murrah buffaloes were frozen in pellets and straws at a final sperm concentration of 80 × 106 mL. We investigated the effect of supplementations on the motility, morphology of spermatozoa at 1 h and 2 h after thawing, sperm plasma membrane status and mitochondrial trans-membrane potential. The comparative CASA analysis of the motility and velocity parameters of spermatozoa showed significant differences in the progressive motility and in the VCL values in favour of control vs. samples with additives (p < 0.05). The molecular changes in PM phospholipid asymmetry were higher after thawing (p < 0.05). The semen treated with Vit C showed significant (p < 0.05) lower number of apoptotic spermatozoa (6CF+/Ann V Cy3+) in comparison with samples incubated with ME. The numbers of dead spermatozoa (6CF-/Ann V Cy3+) in samples with ME were significantly higher than other groups. The percentage of spermatozoa with well preserved and functioning mitochondria in control semen was higher (p < 0.05) than that in the experimental group with ME. The results obtained revealed that addition of Vit C (5 µmol/L) and ME (50 µmol/L) has no positive effect on post-thaw semen quality of buffalo sperm, because of the impaired integrity of PM and mitochondrial trans-membrane potential.

Myoinositol Supplementation of Freezing Medium Improves the Quality-Related Parameters of Dog Sperm.

Simple SummaryThe generation of free radical reactive oxygen species during freeze–thaw procedures is one of the major factors affecting the function and survival of sperm. Myoinositol is the most important natural form of inositol produced in the human body. Researchers have attempted to exploit the antioxidant nature of myoinositol to treat human infertility issues via the improvement of sperm quality traits and fertilization rates. We investigated the potential role of myoinositol neutralizing free radicals produced during the cryopreservation of dog semen. Myoinositol supplementation in the freezing medium resulted in improved quality-related parameters of dog semen including percentage motility, viability, plasma membrane integrity, and chromatin integrity. Improvement in post-thaw semen quality was confirmed by the expression of genes related to apoptosis, nuclear integrity, and reactive oxygen species generation. Oxidative stress during freeze–thaw procedures results in reduced semen fertility. A decrease in free radical levels can improve the post-thaw sperm quality. We examined the effects of myoinositol supplementation in freezing medium on the structure and function of cryopreserved dog sperm. Pooled ejaculates were diluted with buffer without or with myoinositol (1 or 2 mg/mL). Analysis of fresh semen revealed that the optimal concentration of myoinositol was 1 mg/mL, and this concentration was used in further experiments. Post-thaw semen quality in the myoinositol-supplemented group was superior (p < 0.05) compared with that in the control group in terms of motility (57.9 ± 0.4% vs. 47.8 ± 0.2%), sperm viability (57.5 ± 0.5% vs. 44.6 ± 0.6%), intact plasma membrane (56.6 ± 0.4% vs. 46.2 ± 0.6%), and acrosome membrane (59.3 ± 0.5% vs. 51.8 ± 0.5%). In addition, sperm in the myoinositol-supplemented group showed a significantly lower expression of pro-apoptotic (BAX) and mitochondrial reactive oxygen species (ROS) modulator (ROMO1) genes but higher expression of anti-apoptotic (BCL2), and protamine-related (PRM2 and PRM3) genes compared with that in the control group. Therefore, myoinositol supplementation before freezing can protect against oxidative stress and improve post-thaw dog sperm quality.

Bovine sperm selection procedure prior to cryopreservation for improvement of post-thawed semen quality and fertility

BackgroundThe application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction. However, a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation, rendering the frozen-thawed semen impractical for routine use. Cryopreservation is known to reduce sperm lifespan and fertility. Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination (AI) centers. Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process.ResultsOur strategy of sperm selection based on rheotaxis and thermotaxis (SSRT) on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity, viability and biological function related to fertilization competence. This strategy has a positive effect on the cryosurvival and fertilizing abilities of spermatozoa as supported by the improvement on pregnancy rate of Japanese Black heifers and Holstein repeat breeders. This technique protected further sublethal damage to bovine spermatozoa (higher % cryosurvival than the control) and resulted in the improvement of DNA integrity. Prefreeze selected spermatozoa demonstrated slower and controlled capacitation than unprocessed control which is thought to be related to sperm longevity and consequently to appropriate timing during in vivo fertilization.ConclusionsThese results provide solid evidence that improvement of post-thawed semen quality by SSRT method is beneficial in terms of cryosurvival, longevity of post-thawed sperm, and optimization of in vivo fertilization, embryo development and calving as supported by the favorable results of field fertility study.

Improved Post-Thaw Quality of Canine Semen after Treatment with Exosomes from Conditioned Medium of Adipose-Derived Mesenchymal Stem Cells.

Simple SummaryIn this study, we have tried to exploit the potential role of exosomes-derived from canine adipose-derived mesenchymal stem cells in the protection and repair of damaged sperm during cryopreservation. The treatment of sperm with an optimal level of exosomes resulted in improved post-thaw semen quality. This improvement was due to the exosomal proteins that protect the sperm against oxidative damage and induce the repair of membranes and chromatin. The effect of exosomal proteins was also confirmed by higher expression of genes related to the repair of membranes and chromatin accompanied by the lower expression of a gene associated with reactive oxygen species production.Freezing decreases sperm quality, ultimately affecting fertilizing ability. The repair of freeze-damaged sperm is considered crucial for improving post-thaw viability and fertility. We investigated the effects of exosomes derived from canine adipose-derived mesenchymal stem cells on dog sperm structure and function during cryopreservation. The pooled ejaculate was diluted with buffer, without (Control), or with exosomal proteins (25, 50, or 100 µg/mL). Using fresh semen, the determined optimal exosomal protein concentration was 50 µg/mL (Group 2) which was used in further experiments. Post-thaw sperm treated with exosomes were superior to control (p < 0.05) in terms of motility (56.8 ± 0.3% vs. 47.2 ± 0.3%), live sperm percentage (55.9 ± 0.4% vs. 45.4 ± 0.4%), membrane integrity (55.6 ± 0.5% vs. 47.8 ± 0.3%), and acrosome integrity (60.4 ± 1.1% vs. 48.6 ± 0.4%). Moreover, expression of genes related to the repair of the plasma membrane (ANX 1, FN 1, and DYSF), and chromatin material (H3, and HMGB 1) was statistically higher in exosome-treated sperm than control, but the expression of the mitochondrial reactive oxygen species modulator 1 gene was significantly higher in control. Therefore, exosomal treatment may improve the quality of post-thaw dog semen through initiating damaged sperm repair and decreasing reactive oxygen species production.

Comparative Evaluation of Motility and Kinematics of Fresh Versus Frozen-Thawed Spermatozoa of Cattle and Buffalo Bull by CASA

A study was undertaken on semen of three mature bulls each of Gir, Surti and Murrah breed to evaluate the comparative motion characteristics and kinematics of their fresh and frozen-thawed spermatozoa by Biovis CASA. The ejaculates (n= 24/breed) having &gt;75% initial motility were diluted @ 80 million sperm/ml using TFYG extender and were assessed. Amongst motility traits, the total motile, rapid progressive motile and slow progressive motile spermatozoa percentage decreased significantly by 23.08 - 30.09, 43.57 - 55.18, 9.12 - 22.75 %, respectively, plessthan0.01), while non-progressive motile sperm (4.78 - 21.48%) and immotile sperm (164.38 - 178.38 %) percentage increased significantly ( plessthan0.01) in frozen-thawed semen compared to that of fresh semen. The post-thaw quality of semen of all three breeds was in acceptable range. The mean values of sperm velocity/kinematic parameters observed in frozen-thawed semen of Gir, Surti and Murrah bulls, based on total motile sperms, viz., average path velocity, curvilinear velocity, straight line velocity, linearity, straightness, beat-cross frequency, amplitude of lateral head displacement, dancing frequency and dancing mean decreased significantly by 13.70 - 17.79; 9.76 - 12.95; 13.28 - 21.90; 7.28 - 9.68; 4.36 - 7.79; 15.56 - 25.15; 8.78 - 10.50; 6.16 - 18.67 and 12.98 - 15.96 %, respectively, as compared to that of their fresh semen samples. However, wobbling index remained almost same for both fresh and frozen semen. All motility traits differed but none of kinematics/velocity traits differed significantly between breeds/species. The values of all velocity parameters for progressive motile sperms were higher than total motile sperms in all three breeds. The effect of freezing-thawing on velocity and kinematic attributes was much less compared to absolute sperm motility, and both the fresh and frozen-thawed sperms behaved identically with respect to their velocity and kinematics. The rapid progressive motile sperm in both fresh (r=0.41 to 0.92) and frozen-thawed (r=044 to 0.88) semen had significant correlations with most of their velocity traits, and the later were significantly and positively or negatively inter-related among each other in semen of all three breeds. It was therefore concluded that cryopreservation process significantly reduces the motility and kinematics attributes of bovine spermatozoa and, CASA analysis of fresh semen for motility and velocity traits could predict the post-thawed sperm motility and velocity/ kinematics of spermatozoa.

Ameliorative Effect of Ascorbic Acid and Glutathione in Combating the Cryoinjuries During Cryopreservation of Exotic Jack Semen

The present study was designed to study the adverse effects of cryopreservation and evaluation of the cryoprotective effect of reduced glutathione (GSH) and ascorbic acid (AA) supplementation on exotic jack semen in combination or alone. For this, 24 semen samples from four adult and fertile jacks were collected via artificial vagina using an estrus jenny as dummy. After semen collection, the semen was evaluated for various qualitative and quantitative parameters in fresh, cooled, and frozen-thawed semen. The semen pellet was extended with the freezing extender containing either AA (0.9 g/L), GSH (2.5 mM), or combination of both (AA 0.9 g/L + GSH 2.5 mM), and another aliquot was kept as control without adding the antioxidants. The jack semen underwent cryodamage, which was evident by the observation of significant (P < .05) decline in the seminal quantitative parameters at various stages of cryopreservation process. Prefreeze and postthaw semen evaluation revealed that the values of plasma membrane, acrosome integrity, and chromatin integrity were found to be significantly higher (P < .01) in the group of samples supplemented with the combination (0.9 g/L AA +2.5 mM GSH) than AA- and GSH-alone or control groups. Supplementation of antioxidants to the freezing extender improved jack prefreeze and postthaw semen quality with the superiority of GSH over AA alone. From the present study, it was inferred that, exotic jack spermatozoa are susceptible to injuries because of cryopreservation, but these cryo-induced damage can be ameliorated significantly (P < .05) with the use of antioxidants and contribute to the improvement of semen cryopreservation procedures.

Semen cryopreservation for the Mediterranean brown trout of the Biferno River (Molise-Italy): comparative study on the effects of basic extenders and cryoprotectants

This study was designed to optimize the semen freezing protocol of the native Mediterranean brown trout inhabiting the Molise rivers through two experiments: an in vitro analysis of the effects of two basic extenders combined with three cryoprotectants on post-thaw semen quality; and an in vivo test to assess the fertilization and hatching rate. Semen was diluted at a ratio of 1:3 in a freezing medium composed of a glucose extender (A) or mineral extender (B). Each basic component contained 10% dimethylsulfoxide, dimethylacetamide or methanol. The post-semen quality was evaluated considering motility, duration of motility, viability and DNA integrity. The basic extender and cryoprotectant were shown to have significant effects on these variables, and the best results were obtained using extender A or B combined with dimethylsulfoxide (P < 0.05). These freezing protocols were selected for fertilization trials in vivo. Fertilization and hatching rates were significantly higher in fresh semen. No significant differences were observed in frozen semen using extender A or B, although higher percentages of eyed eggs and hatching rates were recorded using extender A. According to our in vitro and in vivo results, the glucose-based extender and dimethylsulfoxide emerged as the best combination for an effective cryopreservation protocol for semen of this trout.

How to Increase Post-Thaw Semen Quality in Poor Freezing Stallions: Preliminary Results of the Promising Role of Seminal Plasma Added after Thawing.

Simple SummaryApproximately half of all stallions are considered poor freezers, which means that they produce ejaculate with poor cryopreservation tolerance. Although the quality of the frozen–thawed semen of poor freezing stallions is low, they can carry and transfer desirable traits to future generations via insemination. Therefore, increasing the post-thaw quality of cryopreserved semen from these stallions is an important step in efficient horse breeding. In this study, we aimed to find a way to increase the quality of cryopreserved semen from poor freezing stallions. We studied the effects of different seminal plasma (SP) additions on various functional parameters of spermatozoa. The results show that the addition of different types of seminal plasma positively affects motility and maintains the status of the plasma membrane of poor freezing stallions. Since the percentage of stallions with poor freezability is quite large, future research should target the discovery of specific seminal plasma components that could be used to increase the post-thaw quality of their spermatozoa.The aim of this study was to evaluate the effect of the addition of two types of seminal plasma (SP) after thawing on the functional characteristics of frozen–thawed (F–T) spermatozoa of poor freezing stallions during prolonged incubation periods. Seminal plasma from stallions with 35–40% (standard seminal plasma, (S-SP)) and 60–70% (above standard seminal plasma, (A-SP)) progressively motile spermatozoa after thawing was used. The motility, kinematic parameters (Computer Assisted Sperm Analysis), distribution of spermatozoa into subpopulations, integrity (carboxyfluorescein diacetate/propidium iodide staining), and functionality (hypo-osmotic swelling (HOS) test) of the spermatozoa plasma membrane were evaluated after thawing (T0) and after 30 min (T30) of incubation at 37 °C. There was no effect of SP addition on spermatozoa motility, but there was a significant positive effect on the kinematic parameters at T0 and T30. The addition of SP significantly increased the percentage of spermatozoa in the fast subpopulation at T0 as well as at T30. Plasma membrane integrity was not affected by the treatment, but functionality significantly decreased by 5% compared to the control group when samples were incubated for 30 min with A-SP. In conclusion, generally, the post-thaw addition of seminal plasma positively affected the post-thaw quality of semen from poor freezing stallions.

Effect of Fractionated Seminal Plasma on Sperm Characteristics Following Cryopreservation of Boar Semen

Abstract This study aimed to investigate the effect of fractionated seminal plasma (SP) on boar sperm characteristics following cryopreservation. Gel filtration chromatography yielded two fractions: SP1 with more than 40 kDa (&gt;40 kDa) and SP2 with less than 40 kDa (&lt;40 kDa). The fractionated SP (SP1 and SP2), whole seminal plasma (wSP) and Beltsville Thawing Solution (BTS) were used for the treatment of semen before freezing-thawing. Besides the analysis of sperm motility characteristics, plasma membrane integrity (PMI), acrosome integrity, and viability (Vybrant Apoptosis Assay) were analyzed in pre-freeze and post-thaw (PT) semen. Among the analyzed pre-freeze sperm parameters, rapid movement was markedly affected by boar and treatment. Furthermore, boar and treatment were significant sources of variations in PT semen quality. Treatment with wSP caused a marked reduction in PT semen quality compared with BTS, SP1 or SP2. Wide variations in PT acrosome integrity and viability were observed in spermatozoa treated with BTS and the fractionated SP, being significantly higher in the SP1- and SP2-treated samples. However, PT semen quality did not differ between semen samples treated with SP1 and SP2. Representative electrophoretic profiles of sperm proteins from each treatment showed quantitative and qualitative differences, indicating varying effects of the cryopreservation procedure on the sperm membrane integrity. The findings of this study indicated that the cryoprotective effects of the SP components varied among boars and that different components of the fractionated SP exerted varying effects on sperm functions following cryopreservation. It could be suggested that the variable protective protein components of either fractionated SP ameliorated alterations in the sperm membranes during cryopreservation, resulting in reduced susceptibility to cryo-damage.

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

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

Optimization of Sperm Cryopreservation Protocol for Mediterranean Brown Trout: A Comparative Study of Non-Permeating Cryoprotectants and Thawing Rates In Vitro and In Vivo

Simple SummaryCryobanking is an important tool to preserve the genetic resources of fish species. Semen cryopreservation has been extensively used in conservation programs for endangered species. Here, we aimed to find an effective cryopreservation protocol for the autochthonous Mediterranean brown trout inhabiting the Biferno river (south Italy), in order to create a sperm cryobank. Low-density lipoproteins and sucrose were tested as non-permeating cryoprotectants (NP-CPAs) to replace the egg yolk. Moreover, the thawing rate (10 °C for 30 s vs. 30 °C for 10 s) was also studied. From results obtained in vitro and in vivo, egg yolk emerged as the best NP-CPA and the lower thawing rate recorded better post-thaw semen quality in vitro and higher fertilization and hatching rates in vivo. These findings are important because they will contribute to the creation of a sperm cryobank for Molise’s native trout, which is a milestone of our European project (Life Nat.Sal.Mo).The aim of our study was to test the effects of different non-permeating cryoprotectants (NP-CPAs), namely low-density lipoproteins (LDLs), sucrose, and egg yolk, and thawing rates on the post-thaw semen quality and fertilizing ability of the native Mediterranean brown trout. Pooled semen samples were diluted 1:3 (v:v) with 2.5%, 5%, 10%, or 15% LDL; 0.05, 0.1, or 0.3 M sucrose; or 10% egg yolk. At the moment of analysis, semen was thawed at 30 °C/10 s or 10 °C/30 s. The post-thaw semen quality was evaluated, considering motility, the duration of motility, viability, and DNA integrity. Significantly higher values of motility and viability were obtained using egg yolk/10 °C for 30 s, across all treatments. However, LDL and sucrose concentrations affected sperm cryosurvival, showing the highest post-thaw sperm quality at 5% LDL and 0.1 M sucrose. Based on the in vitro data, egg yolk, 5% LDL, and 0.1 M sucrose thawed at 10 °C or 30 °C were tested for the in vivo trial. The highest fertilization and hatching rates were recorded using egg yolk/10 °C (p < 0.05). According to these in vitro and in vivo results, egg yolk emerged as the most suitable NP-CPA and 10 °C/30 s as the best thawing rate for the cryopreservation of this trout sperm, under our experimental conditions.

Identifikasi Bakteri dan Efektivitas Antibiotik dalam Pengencer untuk Mengontrol Pertumbuhan Bakteri pada Semen Sapi Friesian Holstein

This study was designed to investigate the presence of bacterial species in Friesian Holstein (FH) bovine semen at the time of collection, processing and to assess the efficacy of two types of antibiotics combinations; penicillin and streptomycin (PS) and gentamycin, tylosin, lincomycin and spectinomycin (GTLS) in semen extender on bacterial control and quality of semen. For this purpose, three experiments were conducted. In experiment 1, identification of bacterial content in fresh semen which collected from 5 bovine ejaculates. In experiment 2, identification of bacterial content in skimmilk-eggyolk extender which were prepared in artificial insemination center, Lembang, Bandung. In experiment 3, identification of bacterial content in frozen thawed semen. In the result, some of bacterial species were isolated from the bovine semen. The GTLS combination of antibiotics may be incorporated into a freezing extender or protocol without compromising the post-thawed semen quality of FH bull spermatozoa. Three types of bacteria were found in fresh semen; Klebsiella sp., Micrococcus sp., and Pediococcus sp.. Three types of bacteria were found in semen extender; Enterobacter cloacae, Pseudomonas diminuta and Serratia plymutica. Two types of bacteria were found in frozen semen; Enterobacter cloacae and Serratia plymutica. In conclusion, antibiotics PS and GTLS were effective for controlling the growth of bacteria in frozen semen.

Stimulating effects of Quercetin (QUE) in tris citric acid extender on post thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa

Aim of this study was to evaluate the stimulating effects of quercetin (QUE) on post thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa. QUE is a well-known flavonoid having antioxidant properties to scavenge free radicals. Semen was collected from three buffalo bull through artificial vagina (42 °C) and were evaluated initially for volume, motility, concentration, followed by dilution in five extenders (n = 5 aliquots, control, C = no QUE; treatment 1, T1 = 50 μM QUE; treatment 2, T2 = 100 μM QUE; treatment 3, T3 = 150 μM QUE and treatment 4, T4 = 200 μM QUE supplementation). The experiment was repeated thrice (n = 3 replicates). At post dilution, sperm progressive motility (PM, %) plasma membrane integrity (PMI, %), supra vital plasma membrane integrity (SVPMI, %) and acrosome integrity (ACR-1, %) of buffalo bull were significantly higher (P < 0.05) in extenders supplemented with QUE than C. At thawing, sperm PM of buffalo bull was higher in T3 and T4 than C, T1 and T2. Sperm SVPMI was significantly higher in T2, T3 and T4 than C and T1. Sperm ACR-I was higher in T3 and T4 than C, T1 and T2. Sperm DNA integrity was higher in T4 than C. QUE supplementation increased in vitro semen quality during 30 and 60 min of incubation at 37 °C than C. The in vivo fertility was higher in T3 and T4 than C (%, 61.82 vs. 65.22 vs. 46.90). It is concluded that QUE @ 150 and 200 μM improved post thaw semen quality and in vivo fertility of buffalo bull.

Effect of Moringa oleifera leaves extract on post–thawed semen quality of Senduro Goat

The present study was conducted to evaluate the supplementation of Moringa leaves extract in CEP-2 Egg Yolk extender on post-thawed semen quality of Senduro goat semen. Ejaculates were collected once a week for ten weeks from one mature buck using an artificial vagina. CEP-2 egg yolk extender containing a various concentration of Moringa leaves extract (1,3,5,7 %) was prepared in mini French straw (0.25 mL). Semen quality was assessed based on post-thawed sperm motility, viability, abnormality and plasma membrane integrity. The result showed that supplementation of different concentration of Moringa leaves extract in CEP-2 Egg Yolk extender have a significant effect (P<0.05) on the percentage of sperm motility, viability, and membrane integrity, but there was no significant effect on the percentage of abnormality (P>0.05). In conclusion, the addition of Moringa oleifera leaves extract 5 % to semen extender could improve post-thawed semen quality.