Proportion Of Spermatozoa Research Articles

Subtle membrane changes in cryopreserved bull spermatozoa when modified temperature drop rates are used during the first phase of freezing

BACKGROUND: Cryopreservation of spermatozoa involves reduction of temperature to a subzero level, leading to increased longevity. However, temperature reduction has a significant effect on sperm membranes. OBJECTIVE: To evaluate the impact of the rate of temperature drop during the first phase of freezing on subtle membrane changes in cryopreserved bull spermatozoa. MATERIALS AND METHODS: Thirty-two ejaculates from four bulls (eight ejaculates/bull) were collected using artificial vagina while keeping a 3 to 4 days gap between two collections. Diluted semen samples were equilibrated at 5°C for 4 hours. The samples were then placed in a pre-programmed semen freezer. The first phase of freezing, that is, 5°C till −10°C was subjected to three different temperature drop rates: accelerated (F1), moderate (F2), and slow (F3), at 20°C per min, 10°C per min and 5°C per min, respectively. After thawing, spermatozoa were assessed for percentage live, plasma, and acrosomal membrane integrity, along with the external appearance of phosphatidyl serine, indicating apoptosis. RESULTS: A significant difference (p<0.05) in viability, plasma membrane integrity (HOS test), and acrosome membrane integrity (PSA test) was observed between F3 and the other groups. However, the parameters did not significantly differ between F1 and F2. The annexin V-PI assay (AN/PI) categorized four types of sperm populations: non-apoptotic and viable (AN−/PI−), apoptotic and viable (AN+/PI−), non-apoptotic and non-viable (AN−/PI+), and apoptotic and non-viable (AN+/PI+). The proportion of spermatozoa with (AN−/PI−) and (AN+/PI+) differed significantly (p<0.05) between F3 and the other groups. The values for apoptotic and viable (AN+/PI−) and non-apoptotic and non-viable (AN−/PI+) sperm were not significantly different among all freezing categories. CONCLUSION: A slower temperature drop rate (freezing rate) during the first phase of freezing results in less damaging, subtle membrane changes.

Quality and Proportion of Spermatozoa in the Percoll Density Gradient Centrifugation Sexing Method at Different Gradients using AndroMed® Diluent

The utilization of sexing technology is an effort made to improve the efficiency of livestock farming, which was created to predict the sex of the calves born so that it can be adjusted to the objectives of the farm. This study aims to analyze the quality and proportion of sexed spermatozoa using the percoll density centrifugation method at different gradients with AndroMed® diluent. The research material used was fresh semen from Belgian Blue Bull aged six years with a body weight of 600 kg and fresh semen quality motility ≥70%. This research is a laboratory experiment with two treatments and ten replicates. The treatments in the study were T0: Ten Gradient density percoll + AndroMed® and T1: Five Gradient density percoll + AndroMed®. The research analysis used a dependent t-test. The statistical analysis showed a significant difference (P<0.05) in treating ten gradients with five gradients in each layer regarding individual motility, viability, abnormality, and total motile spermatozoa. At the same time, in concentration, there was no difference (P>0.05) in the treatment of 10 gradients with five gradients. The average results on individual motility, abnormality, concentration, and total motile spermatozoa showed treatment of 10 gradients better than treatment of 5 gradients in each layer. At the same time, the variable viability showed that the gradient treatment is better than the five-gradient treatment. Proportion of spermatozoa in the Upper Layer T0 X (23.4%), Y (76.8%) and T1 X (16.9%), Y (83.1%). The proportion of Spermatozoa in the Bottom Layer was T0 X (84.1%), Y (15.9%), and T1 X (83%), Y (17%), respectively. In conclusion, sexing spermatozoa with the Percoll 10 and 5 Gradient Density Centrifugation method can separate X and Y spermatozoa.

296 Comparison of Dna Methylation Patterns in Sperm Collected from Brahman Bulls with Differences in Embryo Development

Abstract The objective was to determine if methylation patterns in sperm differed between bulls with good vs poor embryo development. Semen collected from sexually mature Brahman bulls was purified from frozen-thawed straws using a gradient [50% (vol/vol) and 90% (vol/vol)] of Isolate (Irvine Scientific, Santa Ana, CA), and used to fertilize abattoir-derived cumulus oocyte complexes (n = 81-100/bull in 2 wells). Number of blastocysts were recorded on d 8, and bulls were classified as Good (GD; >35% blastocyst rate; n = 5) or Poor (PD; < 35% blastocyst rate; n = 4) embryo development. Methylation of sperm DNA was assessed by reduced representation sodium bisulfite sequencing. Differences in methylation were determined with the Dispersion Shrinkage for Sequencing method in conjunction with Wald test. Differentially methylated cytosines and regions located in promoters of genes and gene bodies were analyzed to reveal gene ontology terms associated with them. Proportion of sperm cells that were viable (before and after a three-hour stress test), with inceased reactive oxygen species (ROS), and with damaged chromatin was determined. Statistical analysis (PROC GLIMMIX; SAS 9.4) included the fixed effect of embryo development classification which was considered significant at P < 0.05. Mean separation was performed using LSmeans. The proportion of viable (P > 0.21; GD = 46 ± 5%; PD = 36 ± 5%) sperm cells did not differ before the stress test. Post-stress test, PD bulls had more viable sperm cells (P < 0.02; 37 ± 3%) than GD (25 ± 2%) bulls. There was no difference between GD and PD for ROS (H2O2; P > 0.16 and •O2; P > 0.59). Proportion of sperm cells with damaged chromatin was greater (P < 0.01) in PD (3 ± 0.3%) than GD bulls (1 ± 0.01%). There were 1,742 differentially methylated cytosines and 343 differentially methylated regions when GD bulls were compared with PD bulls. Genes whose promoters were differentially methylated included DDC, GTSF1L, PPP1R27, NTM, HCK, GPR173, and TNFRSF1A. Functionally enriched pathways for differentially methylated promoters included apoptosis and amyotrophic lateral sclerosis while some of the significant biological processes were regulation of apoptotic process involved in morphogenesis, negative regulation of neuron migration, pyrimidine nucleoside catabolic process, and regulation of cardiac muscle tissue development. Differentially methylated gene bodies were significantly related to cellular components such as cell surface, endomembrane system, synapse, and cell periphery while molecular functions included binding, protein binding, and transmembrane receptor protein tyrosine kinase activity. Biological processes impacted by differentially methylated gene bodies included system development, neurogenesis, animal organ development, anatomical structure development, generation of neurons, nervous system development, and multicellular organism development. In summary, differences in embryo development are likely due to differences in methylation patterns impacting developmental processes, as semen characteristics were mainly similar between bulls with good versus poor embryo development.

Effects of preservation period on quality of chilled semen of Boer-cross bucks

Characteristics of fresh semen and the effect of chilling on the quality of semen of Boer-cross bucks were studied. Fresh semen from two Boer-cross bucks was evaluated for volume, mass motility, progressive motility, viability, concentration and proportion of spermatozoa with normal acrosome, mid-piece and tail. The semen was diluted at 1:4 ratios with Tris-fructose-citrate-egg yolk extender, preserved at 4ºC for 72 hours and evaluated for progressive motility and proportion of spermatozoa with normal acrosome, mid-piece and tail at 24-hour intervals for three days. The mean volume, mass motility, progressive motility, sperm viability, sperm concentration and proportion of spermatozoa with normal acrosome, mid-piece and tail of fresh semen of Buck 1 and Buck 2 were 370.0 ± 0.1 and 410.0 ± 0.1 μl, 3.3 ± 0.4 and 3.4 ± 0.1, 74.0 ± 13.9 and 76.0 ± 6.5%, 82.3 ± 7.5 and 84.3 ± 4.2%, 2730.0 ± 740.9 × 106/ml and 2584.0 ± 470.1 × 106/ml, and 93.3 ± 2.9 and 94.9 ± 0.4%, respectively. The progressive motility in chilled semen of Buck 1 at days 1, 2 and 3 was 61.2 ± 7.3, 51.0 ± 7.3 and 39.0 ± 5.5%, respectively; in Buck 2, it was 60.6 ± 6.8, 50.2 ± 6.2 and 39.2 ± 5.4%, respectively. The difference in semen parameters among days of preservation was significant (P<0.05) in both bucks. The proportion of spermatozoa with normal acrosome, mid-piece and tail in chilled semen of Buck 1 at days 1, 2 and 3 was 88.0 ± 7.0, 88.8 ± 5.9 and 87.1 ± 7.7%, respectively; in Buck 2, it was 86.6 ± 4.8, 84.1 ± 4.6 and 81.8 ± 4.9%, respectively. It is suggested that the characteristics of fresh semen of these two Boer-cross bucks were within normal range. The quality of semen of Boer-cross bucks deteriorated with chilling up to 72 hours. Improvement of the preservation protocol is required to obtain satisfactory quality of chilled semen of Boer-cross bucks.
 Bangl. vet. 2022. Vol. 39, No. 1-2, 34–42

The Effect of Mycotoxins and Their Mixtures on Bovine Spermatozoa Characteristics.

There is growing concern about the effects of mycotoxins on mammalian reproduction. Although the effects of single mycotoxins have been well documented, the impact of their mixtures on spermatozoon quality is less known. Here, frozen-thawed semen (n = 6 bulls) was in-vitro-cultured (2 h) without (control) or with (i) a single mycotoxin [zearalenone (ZEN), ochratoxin A (OTA), toxin 2 (T2), and diacetoxyscirpenol (DAS)] in a dose-response manner; (ii) binary mixtures (OTA + T2, OTA + ZEN, OTA + DAS, ZEN + T2, DAS + T2 and ZEN + DAS); or (iii) ternary mixtures (OTA + DAS + T2, OTA + ZEN + T2, and ZEN + DAS + T2). Then, the spermatozoa quality was characterized according to its plasma- and acrosome-membrane integrity, mitochondrial membrane potential, and oxidation status by a flow cytometer. Exposure to single mycotoxins or binary mixtures did not affect the spermatozoa characteristics. However, exposure to the ternary mixtures, OTA + DAS + T2 and OTA + ZEN + T2, reduced (p < 0.05) the mitochondrial membrane potential relative to the control. In addition, OTA + ZEN + T2 increased (p < 0.05) the proportion of spermatozoa with reactive oxygen species relative to the control. The most suggested interaction effect between the mycotoxins was found to be an additive one. A synergistic interaction, mainly regarding the oxidation status of the spermatozoa, was also found between the mycotoxins. The current study sheds light on the potential risk of exposing spermatozoa to a mycotoxin mixture.

Ameliorative effect of chitosan nanoparticles in capacitation media on post-thawing in vitro fertilizing ability of bovine spermatozoa.

This study aimed to investigate the effect of supplementation of chitosan nanoparticles (CSNPs) on the capacitation of bovine spermatozoa during the in vitro fertilization process. Hyperactivated motility (HAM) and acrosome reaction (AR) of sperm cells as well as in vitro fertilization and cleavage rates are the main parameters used to estimate the effect of CSNPs on bovine spermatozoa's fertilizing ability. In this study, three different concentrations of CSNPs (10, 20 and 100 μg/mL) were prepared and characterized. Motile spermatozoa were separated from frozen-thawed semen by a swim-up technique and capacitated in Sperm-TALP medium supplemented with heparin only without CSNPs treatment (positive control), heparin + 10 μg/mL CSNPs, heparin + 20 μg/mL CSNPs, heparin + 100 μg/mL CSNPs and the last one served as a negative control tube which supplemented with 10 μg/mL CSNPs without adding heparin. Sperm cells were incubated for 90 min at 39°C in a 5% CO2 incubator and evaluated every 30 min at intervals. Cumulus oophorus complex (COCs) were matured in a 5% CO2 incubator at 39°C and inseminated in vitro with frozen-thawed bull sperm of the above concentrations. The inseminated oocytes were incubated at 39°C in a 5% CO2 incubator for 24 h and then examined for evidence of fertilization. The results of this investigation showed that HAM and AR were best affected by CSNPs at a concentration of 20 μg/mL during an incubation time of 60 min. As time went on, the overall proportion of spermatozoa with progressive motility (PM) decreased across all groups, and a substantially lower value was found at the dose mentioned above. Additionally, the impact of sperm treated with CSNPs on fertilization rate was assessed. The outcomes demonstrated that in comparison to the other concentrations (10 and 100 μg/mL), the positive control and the negative control, the proportion of fertilized oocytes was significantly higher in the CSNPs concentration (20 μg/mL). In conclusion, it could be inferred from this investigation that CSNPs support sperm functions during IVF and can be used for biomedical interventions in bovine spermatozoa. Additionally, a high IVF rate was achieved by using sperm treated with CSNPs as CSNPs enhance sperm capacitation and acrosome reaction.

Long-term exposure to aryl hydrocarbon receptor agonist neburon induces reproductive toxicity in male zebrafish (Danio rerio)

Neburon is a phenylurea herbicide that is widely used worldwide, but its toxicity is poorly studied. In our previous study, we found that neburon has strong aryl hydrocarbon receptor (AhR) agonist activity, but whether it causes reproductive toxicity is not clear. In the present study, zebrafish were conducted as a model organism to evaluate whether environmental concentrations of neburon (0.1, 1 and 10 µg/L) induce reproductive disorder in males. After exposure to neburon for 150 days from embryo to adult, that the average spawning egg number in high concentration group was 106.40, which was significantly lower than 193.00 in control group. This result was mainly due to the abnormal male reproductive behavior caused by abnormal transcription of genes associated with reproductive behavior in the brain, such as secretogranin-2a. The proportions of spermatozoa in the medium and high concentration groups were 82.40% and 83.84%, respectively, which were significantly lower than 89.45% in control group. This result was mainly caused by hormonal disturbances and an increased proportion of apoptotic cells. The hormonal disruption was due to the significant changes in the transcription levels of key genes in the hypothalamus-pituitary-gonadal axis following neburon treatment. Neburon treatment also significantly activated the AhR signaling pathway, causing oxidative stress damage and eventually leading to a significant increase in apoptosis in the exposed group. Together, these data filled the currently more vacant profile of neburon toxicity and might provide information to assess the ecotoxicity of neburon on male reproduction at environmentally relevant concentrations.

Novel interpretation of sperm stress test and morphology for maturity assessment of young Norwegian Red bulls

The use of genomic selection significantly reduces the age of dairy bulls entering semen production compared to progeny testing. The study aimed to identify early indicators that could be used for screening bulls during their performance testing period and could give us insight into their future semen production performance, acceptance for the AI station, and prediction of their future fertility. The study population consisted of 142 young Norwegian Red bulls enrolled at the performance test station, followed until we received semen production data, semen doses, and, subsequently, non-return rates (NR56) from the AI station. A range of semen quality parameters were measured with computer-assisted sperm analysis and flow cytometry from ejaculates collected from 65 bulls (9–13 months). The population morphometry of normal spermatozoa was examined, showing that Norwegian Red bulls at 10 months of age have homogenous sperm morphometry. Norwegian Red bulls could be separated into 3 clusters according to their sperm's reaction patterns to stress test and cryopreservation. Results of semi-automated morphology assessment of young Norwegian Red bulls showed that 42% of bulls rejected for the AI station and 18% of bulls accepted had ejaculates with abnormal morphology scores. For the youngest age group at 10 months, the mean (SD) proportion of spermatozoa with normal morphology was 77.5% (10.6). Using novel interpretation of sperm stress test combined with sperm morphology analysis and consecutive cryopreservation at a young age allowed identification of the candidate's sperm quality status. This could help breeding companies introduce young bulls earlier to the AI stations.

Annual trends in semen parameters among men attending a fertility center between 2016 and 2022

BACKGROUND: Multiple studies have shown sperm concentration and count decline in many countries, however, authors conclusions were inconsistent. These types of studies were not conducted in Russia so far.&#x0D; AIM: To evaluate and analyze semen parameters among men attending a fertility center in St. Petersburg between 2016 and 2022.&#x0D; MATERIALS AND METHODS: In a retrospective study parameters of 14234 consequent semen analyses performed according to WHO guidelines (2010) were sorted, analyzed and compared by year.&#x0D; RESULTS: The distribution parameters of the ejaculate volume did not have change over observation period. Azoospermia and cryptozoospermia were detected in 597 (4.2%) and 435 (3.1%) men respectively. A downward trends for sperm concentration of 1.6 million/ml (1.9%)/year and sperm count of 7 million (1.8%)/year were found, with simultaneous annual increase of 1.3% in the proportion of progressively motile spermatozoa. No noticeable changes in the number of motile spermatozoa and the proportion of spermatozoa with normal morphology were found.&#x0D; CONCLUSIONS: Further research is needed to obtain final conclusions about the gradual decline in the semen quality and reproductive function of men, also in other centers of the Russian Federation.

Alkaline Dilution Alters Sperm Motility in Dairy Goat by Affecting sAC/cAMP/PKA Pathway Activity.

In dairy goat farming, increasing the female kid rate is beneficial to milk production and is, therefore, economically beneficial to farms. Our previous study demonstrated that alkaline incubation enriched the concentration of X-chromosome-bearing sperm; however, the mechanism by which pH affects the motility of X-chromosome-bearing sperm remains unclear. In this study, we explored this mechanism by incubating dairy goat sperm in alkaline dilutions, examining the pattern of changes in sperm internal pH and Ca2+ concentrations and investigating the role of the sAC/cAMP/PKA pathway in influencing sperm motility. The results showed that adding a calcium channel inhibitor during incubation resulted in a concentration-dependent decrease in the proportion of spermatozoa with forward motility, and the sperm sAC protein activity was positively correlated with the calcium ion concentration (r = 0.9972). The total motility activity, proportion of forward motility, and proportion of X-chromosome-bearing sperm decreased (p < 0.05) when cAMP/PKA protease activity was inhibited. Meanwhile, the enrichment of X-chromosome-bearing sperm by pH did not affect the sperm capacitation state. These results indicate that alkaline dilution incubation reduces Ca2+ entry into X-sperm and the motility was slowed down through the sAC/cAMP/PKA signaling pathway, providing a theoretical foundation for further optimization of the sex control method.

The Quality and Proportion of Spermatozoa X and Y in Sexed Frozen Semen Separated with Percoll Density Gradient Centrifugation Method on Friesian Holstein Bull

The Quality and Proportion of Spermatozoa X and Y in Sexed Frozen Semen Separated with Percoll Density Gradient Centrifugation Method on Friesian Holstein Bull

Качественные характеристики замороженно-оттаянного семени (обычное и разделенное по полу ) у быков-производителей голштинской черно-пестрой породы и возраст полового созревания полученных от них телочек

A study was carried out to assess the quality of frozen-thawed semen using the sex separation method and the traditional method in a comparative aspect, and the authors believe that the work will be of some interest to specialists in zootechnical and biological fields, as well as to veterinary specialists. The work was carried out at the Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst and on the basis of LLC “Agrofirma Zarya”, Bogorodsky district, Nizhny Novgorod region in the period from 2017 to 2022. usng the semen of eight sires. To assess the quality of the semen, the index of nuclear DNA fragmentation (nDNA) in chromatin was determined, and the activity of spermatozoa was assessed visually and using the Biola AFS‑500 sperm analyser (ZAO Biola, Moscow). On the basis of the studies conducted, it was found that the morphology of the spermatozoa depends on the individual characteristics of the sires; the content of abnormal spermatozoa varies from 2 to 8% when collected by the traditional method and from 4.10 to 15% when collected by the sexing method. A significant difference between the sires is observed in the iDNA fragmentation index – this indicator varies from 2.66 to 8.62% and from 8.50 to 28.57%, respectively. The division of spermatozoa by sex has a direct effect on the quality indicators, leading to a decrease in their activity. This indicator is 11.2% higher in semen obtained by the traditional method than by the alternative technique. The number of abnormal cells in the semen divided by sex is 2.2% higher than in the semen collected by the traditional method, and the proportion of spermatozoa with fragmented iDNA is 7% higher. The velocity of spermatozoa in the studied doses of semen collected using the technique of sex separation was 96–113 μm/s immediately after thawing, and five hours after incubation at +38°C in a thermostat this indicator decreased to the level of 20–34 μm/s. The age of puberty of their calves obtained from the sexed semen from birth to fruitful insemination is 490 days, which is 16 days more than in group I, where heifers were obtained from the semen frozen in the traditional way (TS). The term of fruiting in heifers born from the use of the sexed semen (SS) was 273 days, while that of heifers born from the use of traditionally frozen semen (TS) was 275 days (2 days more). The age of heifers from birth to first calving in the SS group was 762 days and was 14.4 days more than in the TS group, which was 748 days.

Идентификация популяций клеток крови рыб на основе сверточной нейронной сети для составления лейкограммы

A study was carried out to assess the quality of frozen-thawed semen using the sex separation method and the traditional method in a comparative aspect, and the authors believe that the work will be of some interest to specialists in zootechnical and biological fields, as well as to veterinary specialists. The work was carried out at the Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst and on the basis of LLC “Agrofirma Zarya”, Bogorodsky district, Nizhny Novgorod region in the period from 2017 to 2022. usng the semen of eight sires. To assess the quality of the semen, the index of nuclear DNA fragmentation (nDNA) in chromatin was determined, and the activity of spermatozoa was assessed visually and using the Biola AFS‑500 sperm analyser (ZAO Biola, Moscow). On the basis of the studies conducted, it was found that the morphology of the spermatozoa depends on the individual characteristics of the sires; the content of abnormal spermatozoa varies from 2 to 8% when collected by the traditional method and from 4.10 to 15% when collected by the sexing method. A significant difference between the sires is observed in the iDNA fragmentation index – this indicator varies from 2.66 to 8.62% and from 8.50 to 28.57%, respectively. The division of spermatozoa by sex has a direct effect on the quality indicators, leading to a decrease in their activity. This indicator is 11.2% higher in semen obtained by the traditional method than by the alternative technique. The number of abnormal cells in the semen divided by sex is 2.2% higher than in the semen collected by the traditional method, and the proportion of spermatozoa with fragmented iDNA is 7% higher. The velocity of spermatozoa in the studied doses of semen collected using the technique of sex separation was 96–113 μm/s immediately after thawing, and five hours after incubation at +38°C in a thermostat this indicator decreased to the level of 20–34 μm/s. The age of puberty of their calves obtained from the sexed semen from birth to fruitful insemination is 490 days, which is 16 days more than in group I, where heifers were obtained from the semen frozen in the traditional way (TS). The term of fruiting in heifers born from the use of the sexed semen (SS) was 273 days, while that of heifers born from the use of traditionally frozen semen (TS) was 275 days (2 days more). The age of heifers from birth to first calving in the SS group was 762 days and was 14.4 days more than in the TS group, which was 748 days.

The study of features in sperm and spermatogenesis from males of the genus Ovis with different genotypes

Relevance. Interspecific hybridization of domestic animals with wild related species is considered as one of the promising directions in animal husbandry in the framework of increasing the genetic biodiversity of the gene pool of agricultural animals. The article presents the results of studies of the reproductive characteristics in animals of the genus Ovis with different genotypes.Methods. The objects of research were purebred sheep of the Romanov breed, mouflon and interspecific hybrids from sheep of the Romanov breed with mouflon. The qualitative and quantitative indicators of the sperm at the age of 9, 12 and 18 months were studied. An assessment of the morphometric parameters of spermatozoa from interspecific hybrids is given in comparison with the original parental species. The testes histological studies of purebred and hybrid animals at the age of 12 months were carried out.Results. Differences in several indicators of sperm production and spermatogenesis in purebred and hybrid animals depending on the genotype were revealed. A decrease in the volume of ejaculate and concentration of spermatozoa in hybrid animals relative to purebred males at the age of 12 and 18 months was established in 3.5, 2.6 times and in 1.6, 2.1 times, respectively (р &lt; 0,01). An increase in the proportion of spermatozoa with abnormal morphology in hybrid animals relative to purebred males in 2.9–3.3 times was revealed (р &lt; 0,01). The obtained data are confirmed by histological studies. There is a decrease in the area and volume of seminiferous tubules in hybrid animals by 9.7% and 37.1%, respectively, compared with similar indicators of purebred males of the Romanov breed (р &lt; 0,01). It was shown that in purebred animals in the lumen of the seminiferous tubule have many mature germ cells — sperm, while in hybrid males the presence of single germ cells was established, which indicates a later puberty of hybrid animals compared to the original maternal form — sheep of the Romanov breed.

The efficacy of novel centrifugation-free sperm selection (Io-Lix) on sperm parameters and ICSI reproductive outcomes

Research questionWhat is the effect of a novel non-centrifugation method (Io-Lix) of sperm selection on sperm parameters and intracytoplasmic sperm injection (ICSI) reproductive outcomes? DesignThis pilot study elevated the capacity of the Io-Lix sperm selection protocol to improve sperm parameters (concentration, motility and sperm DNA fragmentation) of the neat ejaculate. Once established, the reproductive outcomes of Io-Lix selected spermatozoa were used for autologous and donor oocyte ICSI programmes and their efficacy compared with those using conventional swim-up. ResultsIo-Lix sperm selection resulted in lower sperm concentration yield (P < 0.001) and sperm DNA fragmentation (P < 0.001) but higher sperm motility (P < 0.001) when compared with spermatozoa in the neat ejaculate. When compared with swim-up sperm selection the Io-Lix protocol resulted in a 14.7% (P = 0.028) increase in pregnancy rate and 16.3% (P = 0.047) reduction in miscarriages in the autologous ICSI programme. A similar comparison of sperm selection procedures employed for a donor oocyte ICSI programme showed no difference in terms of their respective reproductive outcomes. ConclusionsThe Io-Lix sperm selection protocol resulted in improved pregnancy rate and reduction in miscarriage when applied to autologous ICSI, which was attributed to a reduction in the proportion of spermatozoa with DNA damage post-selection. A similar finding was not apparent in the donor oocyte programme, which may be associated with the capacity of the donor oocyte to repair sperm DNA post-syngamy.

Effect of ecotype and age on semen characteristics of three Tanzanian native chickens

Several findings on the semen characteristics of domestic chickens have revealed that ecotype and age significantly affect semen quality. There is, however, lack of data on effects of ecotype and age on semen characteristics of Tanzanian native roosters. This study evaluated the effect of ecotypes (Ching’wekwe, Morogoro-medium and Kuchi) and ages (11-15 and 24-28 months) on semen quality. A total of 192 semen samples were collected from 12 roosters (four from each ecotype) using the abdominal massage technique at weekly interval for four consecutive months. Semen characteristics of individual samples were evaluated. The semen volume, pH, sperm motility, sperm concentration, proportion of spermatozoa with normal morphology and proportion of live spermatozoa among the ecotypes varied from 0.42±0.04 to 0.52±0.03mL, 7.01±0.00 to 7.02±0.00, 72.81±1.27 to 76.63±1.35%, 3.90±0.98 to 4.12±1.96 x 109/mL, 86.16±0.55 to 89.38±0.80% and 88.06±1.13 to 90.97±0.81% respectively. However, only the variations in proportion of spermatozoa with normal morphology and proportion of live spermatozoa among the ecotypes were significant (P&lt;0.05). The semen volume, pH, sperm motility, sperm concentration, proportion of spermatozoa with normal morphology and proportion of live spermatozoa among the two age groups varied from 0.44±0.03 to 0.52±0.03mL, 7.01±0.00 to 7.02±0.00, 73.88±1.13 to75.92±0.99%, 3.80±0.45 to 4.28±0.32 x 109/mL, 87.02±0.58 to 88.15±0.64%, 88.27±0.77 to 89.83±0.77% respectively. However, only the variations in semen volume among the two age groups were significant (P&lt;0.05). The Pearson correlation coefficients between semen volume and other semen quality characteristics were mostly low to medium with positive values ranging from 0.01-0.51 between semen volume and sperm motility and between morphological normal spermatozoa and proportion of live spermatozoa, respectively. Although there is minimal variation in semen quality among ecotypes and age groups, all the ecotypes might still be used in breeding purposes to maintain native chickens, because the results found were within the reference range for chickens.

Effects of fixatives and storage duration on avian sperm morphology

Sperm cells are variable both within and among species. To be able to accurately measure sperm cells and understand their function, it is important that sperm cells are preserved in a manner that maintains their structural integrity. Formalin is a widely used fixative and storage medium for sperm cells, but few studies have examined the effect of fixation and long-term storage on their morphological integrity. Ethanol is also a common fixation and storage agent for tissue samples, and here we examine if fixation and storage in formalin or ethanol alters sperm cell size and structural integrity. We found no significant effects of the fixation process on fresh sperm cells fixed in formalin or ethanol. Further, there were no consistent length changes in sperm cells stored in formalin or ethanol over a period of 227 days, or in sperm cells stored in formalin for three years. A comparison across 13–14 years of storage time showed a small but significant reduction in sperm cell length of 0.93%. Furthermore, sperm cells initially fixed in formalin remained quite stable in dry storage on glass slides for a minimum of six months (we found a mean reduction in sperm cell length of 0.18% after 6 months). The proportion of sperm cells with head damage was, however, much higher for samples stored in ethanol than for those stored in formalin. Overall, 70% of sperm cells had acrosome damage in ethanol versus only 3% in formalin. Finding intact sperm cells for measuring length, therefore, required greater effort in ethanol samples than in formalin samples. Our findings indicate that use of sperm cells from long-term storage for the study of sperm morphometrics is justified for either fixative, although formalin clearly preserves the sperm cells better.

Obesity Surgery Improves Hypogonadism and Sexual Function in Men without Effects in Sperm Quality.

(1) Background: Obesity is associated with hypogonadism, sexual dysfunction, and impaired fertility in men. However, its effects on semen parameters or sexual function remain debatable. (2) Methods: This paper involves a longitudinal study in men submitted for obesity surgery at a university tertiary hospital. Patients were studied at baseline and at 6, 12, and 18 months after obesity surgery. At each visit, anthropometry measures were collected and hormonal and semen parameters were studied. Sexual function was evaluated with the International Index of Erectile Function (IIEF). (3) Results: A total of 12 patients were included. The average body mass index of patients decreased from 42.37 ± 4.44 to 29.6 ± 3.77 kg/m2 at 18 months after surgery (p < 0.05). Hormonal parameters improved after obesity surgery. The proportion of sperm cells with normal morphology tended to decrease from baseline and became most significant at 18 months (5.83 ± 4.50 vs. 2.82 ± 2.08). No significant changes were found in the remaining semen parameters. Erectile function improved significantly at six months after surgery. (4) Conclusions: The authors believe that, in general, the effects of obesity surgery on fertility may be limited or even deleterious (at least in the short and midterm follow-up).

Efficacy of density gradient centrifugation technique (DGC) in enhancing sperm cell DNA quality for assisted reproductive technique

ObjectiveDensity gradient centrifugation (DGC) is commonly used for sperm preparation before assisted reproductive technology (ART) procedures. This technique separates superior motile spermatozoa with normal morphology from the total sperm population. However, there is still controversy as to the effects of this sperm separation technique on sperm cell DNA integrity which is a determining element in the process of fertilization and embryonic development.The objective of this study was to determine the effects of DGC on sperm cell DNA integrity as assessed by a novel association between two cytogenetic tests.Study designsSemen samples were collected from 30 fertile donors and 40 patients being candidates for ART treatment. Each sample was divided into two parts: the first portion was subjected to selection by two layers of DGC (45% and 90%) and the second fraction was rinsed with phosphate-buffered saline solution and centrifuged without density gradient.Abnormal sperm chromatin structure as evaluated by a sperm chromatin dispersion (SCD) test and DNA denaturation as assessed by an acridine orange (AO) test were monitored in the initially washed sample and in the different layers of the density gradient centrifugation.ResultsDGC significantly improved the proportion of sperm progressive motility, total motility, and sperm morphology. Moreover, following density gradient centrifugation, the proportion of spermatozoa with denaturated DNA significantly decreased when compared with whole semen (p < 0.001). In addition, we found that spermatozoa isolated in the 90% layer possessed a significantly lower percentage of sperm chromatin decondensation when compared with those remaining in the 45% layer and unprocessed semen (p < 0.001).ConclusionsUsing double cytogenetic tests, our study shows that semen processing by density gradient centrifugation is useful in selecting sperm with higher double-strand DNA integrity and recommended to be used in sperm preparation for assisted reproduction.

Nanowater enhances cryoprotective properties of glycerol-containing extenders used for ram semen freezing: A preliminary study spanning laboratory testing

Abstract It has been suggested that nanowater (NW-water declustered in the cold plasma generator and characterized by a low freezing point and high diffusivity) could improve ram semen quality after freezing in glycerol-containing extenders. Eighteen ejaculates from six Olkuska rams were divided into six equal portions each, and then diluted (800×106 spermatozoa/ml) and frozen in the fructose-skimmed milk-egg yolk Kareta extenders containing 3% or 7% of glycerol. The extenders were prepared with deionized water (DW-3% and DW-7%) or NW declustered for 15 min (NW15’) or 30 min (NW30’). Post-thaw sperm motility, proportions of sperm defects and percentages of apoptotic, necrotic, and live spermatozoa were determined. The proportion of spermatozoa with midpiece defects was lower (P&lt;0.05) in NW15’-3% compared with DW-3%. Sperm progressive motility was greater (P&lt;0.05) for spermatozoa cryopreserved in both NW30’ (NW30’-3%/7%) extenders compared with their respective controls (DW30’-3%/7%). The proportion of necrotic spermatozoa 1 h after thawing was lower (P&lt;0.05) in NW30’-7% compared with DW-7%, whereas the proportion of live cells detected immediately and 1 h after thawing was greater (P&lt;0.05) in NW30’-7% than in DW-7%. In summary, NW enhanced cryoprotective effects of glycerol-containing extenders with an increase in sperm viability being greater with 7% than 3% of glycerol. Different declustering times appear to alter NW properties. These observations merit future studies of the utility of NW for semen cryopreservation in rams and other mammalian species. The specific mechanisms whereby NW ameliorates the quality of frozen-thawed ram spermatozoa remain to be elucidated.