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The productive longevity of sows leads to an increase in production of marketable products. However, more than half are discarded after the first farrowing due to impaired reproductive qualities under the influence of various factors including infectious diseases. The seminal material of boars can be a source of infection for sows. There is evidence of the relationship between the influence of infectious agents on the reproductive tract of producers and on inflammatory processes in the generative organs of females, which leads to a decrease in reproduction rates. In males, contamination of the sperm-carrying ducts by microorganisms directly affects the quality of sperm production and the function of spermatozoa, which in turn leads to the production of antibodies, reactive oxygen species and DNA fragmentation. To study the effect of bacterial contamination on the biological integrity of sperm in breeding boars, three groups of animals were formed depending on the activity of spermatozoa. For the study, ejaculates of Large White, Landrace and Duroc breeding boars (n= 46) were taken. The age of the producers ranged from 18 to 40 months; ejaculates were collected in JanuaryDecember. To determine qualitative and quantitative parameters of spermatozoa, Argus CASA program (ArgusSoft, St. Petersburg, Russia) was used based on CASA technology, using single-factor analysis methods. A comprehensive assessment of the ejaculates of boar producers showed the presence of yeast and yeast-like fungi (mold) in 44% of the studied samples, in which the total number of colony-forming microorganisms was 489537.5 CFU/cm3, which is 8.1 times more than in samples without contamination, and the proportion of progressive motile spermatozoa was lower by 10%. In the group with high microbial contamination (4895CFU/cm3), activity was 1.2 times lower (p 0.001), the proportion of abnormal spermatozoa was 1.7 times higher (p 0.001) and the level of DNA fragmentation was 3.1 times higher (p 0.001). Correlation analysis between microbial contamination and progressively motile spermatozoa showed a negative bilateral correlationr=0.51 (p 0.01) and a positive correlation with abnormal morphologyr= 0.42 (p 0.05) and DNA fragmentationr= 0.56 (p 0.01). Reproductive indicators of boars-producers in the group with high bacterial contamination were established: fertilizing ability was 23% lower, and the number of abortions was 2.3 times higher, and the frequency of stillbirths and mummified piglets in the litter was 3 times and 2.5 times higher, respectively.

Intracytoplasmic sperm injection (ICSI) is clinically used to treat obstructive/nonobstructive azoospermia. This study compared the efficacy of ICSI with cauda epididymal and testicular sperm in Wistar (WI) and Brown- Norway (BN) rats. The transfer of ICSI oocytes with cryopreserved epididymal and testicular WI sperm resulted in offspring production of 26.2% and 3.7%-4.7%, respectively (P < 0.05). Treatments for artificial oocyte activation (AOA) and acrosome removal improved pronuclear formation in BN-ICSI oocytes; however, only AOA treatment was effective in producing offspring (3.7%-6.5%). In the case of ICSI with testicular sperm (TESE-ICSI), one offspring (0.6%) was derived from the BN-TESE-ICSI oocytes. The application of AOA or a hypo-osmotic sperm suspension did not improve the production of TESE-ICSI offspring. Thus, outbred WI rat offspring can be produced by using ICSI and less efficiently by using TESE-ICSI. Challenges in producing offspring by using ICSI/TESE-ICSI in inbred BN strain require further investigation. [ABSTRACT FROM AUTHOR]

Objective: The sperm sexing using an albumin column requires an incubation process at a certain temperature for sperm to move through the albumin layer. It is thought that the incubation time can influence the quality of the semen resulting from post-thawing sexing. The aim of this research was to determine the effect of incubation time on the quality of sexed sperm post-thawing and to find out the optimum incubation time that provide the best sperm quality. Methods: This research was carried out in a completely randomized design, using three incubation times (T1= 45 minutes, T2= 60 minutes, T3= 75 minutes) with six replications. The parameters consisted of motility, abnormality and acrosome integrity of sexed sperm in the upper and bottom fractions. Data were analyzed using analysis of variance (Anova) and continued with the Duncan test. Results: The results showed that the incubation time had a significant effect on motility, intact acrosome cap and recovery rate, but had no significant effect on abnormality of post thawed sexed sperm. For both the upper and bottom fraction, incubation times of 45 and 60 minutes produced the best motility and recovery rate compared to 75 minutes, but the best intact acrosome cap was obtained from an incubation time of 45 minutes. Conclusions: Based on the research results, it can be concluded that the length of incubation time influences the quality of post thawed ram sexed sperm, and 45 minutes is the optimal incubation time to provide the best quality. [ABSTRACT FROM AUTHOR]

Since the stallion acrosome is very small compared to other species and cannot be properly assessed without additional staining, several labelling techniques were developed to facilitate its assessment. The aim of this study was to compare the Spermac stain (Minitüb GmbH) and a PNA/PSA/PI triple‐staining detected by flow cytometry with regard to method agreement for detecting non‐intact acrosomes within two different extenders. For this purpose, eighteen stallion ejaculates were split in half and diluted with the semen extenders EquiPlus or Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL, respectively. Subsequently, 126 semen samples were stained with both methods between 4 and 240 h (mean: 63.8 ± 48.9 h) after semen collection. Calculated Intraclass correlation coefficients revealed excellent correlations between both methods for EquiPlus (r =.77, p <.001) and fair correlations for Gent (r =.49, p <.001). Interestingly, flow cytometry detected more non‐intact acrosomes in EquiPlus than in Gent (p <.001), whereas the Spermac stain showed no differences (p =.902) between extenders. The poorer method agreement in Gent could be caused by egg yolk artefacts, which made interpretation difficult, so flow cytometry might be preferred. The differences in detected non‐intact acrosomes between extenders highlighted the importance of establishing adapted laboratory protocols for different extender types in order to generate comparable results. [ABSTRACT FROM AUTHOR]

Copyright of Kocatepe Veterinary Journal / Kocatepe Veteriner Dergisi is the property of Afyon Kocatepe University, Faculty of Veterinary Medicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Indonesian Journal of Animal & Veterinary Sciences / Jurnal Ilmu Ternak dan Veteriner is the property of Indonesian Center for Animal Science Research & Development and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The nearly exclusive use of cryopreserved semen in cattle breeding enables long shipping distances, higher storage times, quarantine to avoid germ transmission and easy dispersal of high genetic value bulls. Spermatozoa from bulls are well freezable and improvement of cryopreservation protocols over decades has led to high semen quality. However, there is still some loss of spermatozoa in each semen dose due to detached acrosomes after thawing. There are even individual bulls with extremely high numbers of detached acrosomes after cryopreservation, called "bad freezers". This study screened 1092 ejaculates from 59 Holstein bulls for the difference in detached acrosomes before and after cryopreservation (ΔAC). The individual bull influenced ΔAC (P < 0.001) and allowed selection for individuals with repeatedly low ΔAC (good freezers) or high ΔAC (bad freezers). Good freezers were superior to bad freezers in a thermo-resistance test (78.2% vs. 33.6% total motility, respectively, P = 0.047) and had higher non-return rates (NRR: 46.8% vs. 40.8%, respectively, P = 0.016). Since oxidative stress is one possible explanation for premature acrosome reaction, the radical reduction capacity of the seminal fluid was measured, finding that this parameter was reduced in bad freezer bulls during cryopreservation (P = 0.043). Analysis of lipid species in sperm cells by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed a reduction of ether lipids and plasmalogens as well as an increase in formyl-lysophosphatidylcholines only within the bad freezers during cryopreservation (P = 0.043). In conclusion these findings show, that lipid alteration caused by oxidative stress is one essential reason for highly augmented acrosome reacted spermatozoa in bad freezer bulls. Therefore, increased use of antioxidants in the extender could be a possible starting point for developing individualized extenders for bad freezer bulls of high genetic value, in order to raise sperm quality after cryopreservation even in those bulls. • Bad freezer bulls with high increase of detached acrosomes after cryopreservation. • Cryopreservation reduced radical reduction capacity of seminal fluid. • Ether lipid and plasmalogen depletion in bad freezer bulls by cryopreservation. [ABSTRACT FROM AUTHOR]

The article presents a study on the effect of adding egg yolk to the diluent Androstar Plus for cooling llama semen. It is mentioned that semen cooling is a more viable alternative than deep freezing in South American camelids. The objective of the study was to evaluate the effect of adding egg yolk to the diluent Androstar Plus in cooled llama semen. The results showed that the addition of egg yolk improved semen mobility, but also increased the percentage of dead sperm with damaged acrosomes. Additionally, a decrease in membrane functionality and an increase in tail abnormalities were observed in the cooled samples. [Extracted from the article]

Copyright of Indonesian Journal of Animal & Veterinary Sciences / Jurnal Ilmu Ternak dan Veteriner is the property of Indonesian Center for Animal Science Research & Development and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Growth differentiation factor 9 (GDF9) is a member of the TGFß superfamily that plays a critical role in females' ovarian follicular development and ovulation rate. However, its role in the testis has not been elucidated in Goats. Therefore, this study investigated the effects of GDF9 mutations on the quality of fresh semen in Bucks. Direct sequencing was used to screen possible SNP loci in the goat GDF9 exon one, one SNP locus site 2006 CTC < CTA ( leu < leu ) was identified positively in 15 local Iraqi Bucks. Analysis of variance revealed that significant effect of breeding value for concentration, individual ( p > 0.05) movement, and Acrosome integrity, in All, so of dominance effects of volume, Individual movement, and Acrosome integrity. All traits have low genetic variation, which means that the environment varies high. This study is the first to show a significant association of GDF9 in the initiation or maintenance of spermatogenesis in male goats. [ABSTRACT FROM AUTHOR]

Copyright of Veterinarska Stanica is the property of Croatian Veterinary Institute and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Assisted reproductive technologies have been used in zoos and breeding centres to preserve rare and endangered species of wild animals over the past three decades. The most common way to preserve the genetic material of farm and wildlife animals is to create cryobanks of semen producers and embryos. Studies of domestic species under controlled conditions provide an excellent opportunity to develop effective semen handling techniques for application to wild species of the genus Equus. The All-Russian Research Institute for Horse Breeding (ARRIH) began studies on the cryopreservation of genetic resources in the early 1950s. In 1954 the ARRIH got the world's first foal from the artificial insemination by cryopreserved sperm. The collection of genetic resources of ARRIH has been successfully working since 1972 and contains nowadays cryopreserved sperm from 56 stallions of 16 breeds of domestic and foreign selections. Freezing and subsequent thawing of stallion sperm leads to a decrease in progressive sperm motility and an increase in the number of sperm with structural pathologies. Electron microscopy is one of the most accurate methods used to assess the structural integrity of sperm. We conducted an electron microscopic study of native and cryopreserved sperm of 35 stallions of riding and trotting breeds, aged 4 to 22 years (mean age 11.3 ± 0.9 years). We found that organoids with a denser structure are more resistant to freezing and subsequent thawing of sperm. The use of stallion sperm cryopreservation according to the standard technology of ARRIH leads to a decrease in the number of sperms with intact heads by an average of 19.7%, and the share of sperm with intact heads in cryopreserved sperm is 49.1 ± 2.7% (p < 0.001). The most negatively cryopreservation of semen affects an acrosome of sperms. The main pathologies of acrosomes in sperm cryopreservation are acrosome hypoplasia and its degradation (secondary absence of acrosome). The number of spermatozoa with acrosome hypoplasia and the absence of internal content after cryopreservation increases by 20.9%, and the share of such sperms is 31.7% in thawed sperm (p < 0.001). The number of sperm with acrosome degradation during sperm freezing and thawing increases by 10.4%, and is 18.6 ± 2.4% in average. The share of spermatozoa with normal mitochondrial structure in native sperm is 89.3 ± 2.0% in average, in cryopreserved sperm – decreases by 6.5%. The share of spermatozoa with normal axoneme after cryopreservation decreases slowly, averaging by 4.4% and is 81.4 ± 2.0%. Some of the available assisted reproduction technologies will need to be further optimised in domestic horses before they can be applied to the endangered Przewalski's horse Equus ferus przewalskii.

Summary: A recessive sperm defect of Yorkshire boars was detected more than a decade ago. Affected boars produce ejaculates that contain spermatozoa with defective acrosomes, resulting in low fertility. The acrosome defect was mapped to porcine chromosome 15 but the causal mutation has not been identified. We re‐analyzed microarray‐derived genotypes of affected boars and confirmed that the acrosome defect maps to a 12.24 Mb segment of porcine chromosome 15. To detect the mutation causing defective acrosomes, we sequenced the genomes of two affected and three unaffected boars to an average coverage of 11‐fold. Read depth analysis revealed a 55 kb deletion that is associated with the acrosome defect. The deletion encompasses the BOLL gene encoding the boule homolog, an RNA binding protein which is an evolutionarily conserved member of the DAZ (Deleted in AZoospermia) gene family. Lack of BOLL expression causes spermatogenic arrest and sperm maturation failure in many species. Boars that carry the deletion in the homozygous state produce sperm but their acrosomes are defective, suggesting that lack of porcine BOLL compromises acrosome formation. Our findings warrant further research to investigate the role of BOLL during spermatogenesis and sperm maturation in pigs. [ABSTRACT FROM AUTHOR]

Although useful spermatozoa cryopreservation techniques have been established, long‐term equilibration seems to be required before freezing the spermatozoa of many species, including dogs. The fertility of cryopreserved dog spermatozoa from five males for a reduced equilibration period (0, 30, 60, 120 and 180 min) in a skim milk (SM)‐based extender containing raffinose was evaluated in the present study. When the sperm was diluted with the extender at room temperature (RT) and cryopreserved without equilibration, the proportion of total motile spermatozoa (TMS) after thawing was lower (27%) than when the sperm was equilibrated for 30 min (33%), 60 min (32%), 120 min (44%; p <.05) or 180 min (29%). The proportion of TMS increased as the equilibration time increased and peaked at 120 min. Acrosome integrity was significantly lower in the cryopreserved spermatozoa that had not undergone the initial equilibration than in the equilibrated spermatozoa (p <.05). The normal rate of acrosomes increased with the extension of the first equilibration and peaked at 120 min. When frozen–thawed spermatozoa that had been diluted at RT and subjected to an initial equilibration lasting 60 or 180 min were transcervically inseminated into recipients, there were no differences in the delivery rate, litter size or breeding efficiency. In the cryopreservation of canine spermatozoa using a SM‐based extender, even if the initial equilibration time was shortened to 60 min, the results were comparable to those obtained when the conventional method (with an initial equilibration time of 180 min) was used. [ABSTRACT FROM AUTHOR]

The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single‐layer centrifugation with Androcoll‐E™ (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p ˃.05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies. [ABSTRACT FROM AUTHOR]

The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single‐layer centrifugation with Androcoll‐E™ (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p ˃.05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies. [ABSTRACT FROM AUTHOR]

The wide use of glyphosate-based herbicides (GBHs) has become a matter of concern due to its potential harmful effects on human health, including men fertility. This study sought to investigate, using the pig as a model, the impact of pure glyphosate and its most known commercial formulation, Roundup, on sperm function and survival. With this purpose, fresh commercial semen doses were incubated with different concentrations (0–360 µg/mL) of glyphosate (GLY; exp. 1) or Roundup, at the equivalent GLY concentration (exp. 2), at 38 °C for 3 h. Glyphosate at 360 µg/mL significantly (P < 0.05) decreased sperm motility, viability, mitochondrial activity and acrosome integrity but had no detrimental effect at lower doses. On the other hand, Roundup did significantly (P < 0.05) reduce sperm motility at ≥ 5 µg/mL GLY-equivalent concentration; mitochondrial activity at ≥ 25 µg/mL GLY-equivalent concentration; and sperm viability and acrosome integrity at ≥ 100 µg/mL GLY-equivalent concentration as early as 1 h of incubation. In a similar fashion, GLY and Roundup did not inflict any detrimental effect on sperm DNA integrity. Taken together, these data indicate that, while both glyphosate and Roundup exert a negative impact on male gametes, Roundup is more toxic than its main component, glyphosate. [ABSTRACT FROM AUTHOR]

Egg yolk tris extender is the most appropriate media for cryopreservation in most domestic animals (sperms). In this study we try to discuss that soybean tris extender is more economical and less contaminated to replace the egg yolk tris extender three semen parameters were studied, motility, dead and live and acrosome integrity at zero h, 24h, 48h, 72h after cooling at 5°C. There is significant difference (p<0.05) between (EYT) and 30% concentration of SBT in sperm motility respectively (62.2±1.05, 54.15 ± 1.05), live sperm three concentrations of SBT respectively (71.55 ± 2.85, 60.4 ± 4.0, 70.2 ± 2.5, 63.55 ± 2.7) and acrosome integrity with 30% concentration SBT respectively (84.75 ± 1.25, 73.9 ± 1) after 24 hours of cooling. There is significant difference (p<0.05) between (EYT) and SBT 10%, 30% in sperm motility respectively (45.95 ± 2.15, 38.65±2.5, 41.1 ±2.1). There is significant difference (p<0.05) between (EYT) and SBT 10%, 20%, 30% in sperm livability respectively (71.55±2.85, 60.4 ±4.0, 70.2 ± 2.5, 63.55) and there is significant difference (p<0.05) between (EYT) and SBT 20% in acrosome integrity respectively (77.8 ± 1.25, 80.5 ± 1.3) after 48 hours of cooling. There is significant difference (p<0.05) between (EYT) and SBT 30% in sperm motility respectively (37.3 ± 2.3, 32.15 ± 1.5). There is significant difference (p<0.05) between (EYT) and SBT extender concentrations 10% and 30% in sperm livability respectively (48.9 ± 2.35, 40.3 ± 1.15, 43.4 ± 1.9) and there is significant difference (p<0.05) between (EYT) and SBT extender concentrations 20% respectively (72 ± 1.4, 76.25 ± 1.75) after 72 hours of cooling. Conclusion the SBT concentration 20% is more reliable to replace YET. More research needed to confirm our result. [ABSTRACT FROM AUTHOR]

The present study was aimed to develop a most effective extender for long term storage of Labrador dog semen at 4oC. Tris-citric acid-fructose was supplemented with egg yolk (TEY), egg yolk plasma (TEYP), low density lipoproteins (TLDL) and coconut water (TCW) @10, 15 and 20; 10, 15 and 20; 11-15; and 25, 50 and 75 percent, respectively. Extended semen was stored at 4oC and analyzed for sperm attributes and lipid peroxidation. Values for motility, viability and plasma membrane integrity remained significantly (p < 0.05) higher in 15% TEY, 15% TEYP, 13% TLDL and 25%/50% TCW from 0 hr to 72 hrs of storage. It indicated superiority of 15% TEY, 15% TEYP, 13% TLDL and 25%/50% TCW over other concentrations for storage of Labrador dog semen at 4oC. A significant (p < 0.05) decline in motility, viability and membrane integrity was observed from 0-72 hrs of preservation in all extenders, but sperm attributes were still >50% at 72 hrs of preservation. Decline was comparatively less in 15% TEY, 15% TEYP, 25%/50% TCW and 13% TLDL compared to other concentrations. Values for acrosome integrity also differ significantly (p > 0.05) among different concentrations of extenders except TEY. Lipid peroxidation did not vary among different extenders except TEY. In conclusion, both 15% TEY and 15% TEYP were better than 13% TLDL and 25% TCW. TEY extender may be substituted with TEYP for preservation of dog semen at 4°C and further interventions may improve TLDL and TCW extenders. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the effect of two antioxidants (Trolox and Crocin) added to the freezing extender on the cryopreservation of Pelibuey ram semen. Semen samples were pooled, split and frozen into 3 treatments: T1: Trolox 1mM; T2: Crocin 1mM; TT: Control (without antioxidant). Total motility (MT), progressive motility (PM), sperm viability, mitochondrial activity, acrosomes integrity, and plasma membrane integrity of the tail (Host) were assessed at 0 h (thawed), and at 6 hours at 37 °C. The results were analyzed with ANOVA, followed by Tukey's comparison test. At 0 h and 6 h, no differences were found (P> 0.05) in all the parameters evaluated between treatments, however, from 0 to 6 hours, the MT was similar in the T1 and T2, but not in the TT (P <0.01), on the contrary, acrosome integrity in the three treatments was similar (P>0.05). MP, viability, mitochondrial activity, and Host dropped through time in a similar way in the three treatments. The addition of the antioxidants Trolox and Crocin at 1 mM to the freezing extender allows maintaining the total motility and acrosome integrity during 6 h of incubation at 37 °C. [ABSTRACT FROM AUTHOR]

Background & objectives: The detailed assessment of sperm morphology is important in the semen of infertile men because there is a low proportion of normal spermatozoa. One of the parameters of such sperm morphology is the acrosome, and its effect on assisted reproductive outcomes is controversial. This study was undertaken to evaluate the association between different forms of acrosome on the chromatin status and the assisted reproductive outcomes. Methods: A total of 1587 unstained sperms from 514 infertile men were captured and analyzed for different acrosome forms (normal, large, small, skew, amorphous acrosome and without acrosome) in real time during intracytoplasmic sperm injection into oocytes. The association between the percentage of sperms with atypical acrosome and head shapes and the sperm chromatin status was studied. Fertilization, zygote and embryo quality and clinical pregnancy rates were calculated for different groups of sperms. Results: The highest frequency of irregular shapes of acrosomes, such as small, large and amorphous, was observed in abnormal ellipticity, anteroposterior symmetry and angularity parameters, respectively (P <0.05). The fertilization rate of injected sperms with large (P <0.01) and small (P=0.001) acrosomes and without acrosome (P=0.001) was significantly lower in comparison with normal acrosomes. The quality of zygotes (Z3, P=0.05), embryos (grade C, P <0.05) and the pregnancy rate (P=0.001) from injected sperms with large acrosomes were significantly lower compared with normal acrosomes. Interpretation & conclusions: Our findings showed that the different sperm acrosome morphologies (e.g., large, small, and without acrosome) might negatively relate with chromatin integrity and decrease the sperm's fertility potential and pregnancy rate during intracytoplasmic sperm injection (ICSI) cycles. [ABSTRACT FROM AUTHOR]

The arrangement of immature germ cells changes regularly and periodically along the axis of the seminiferous tubule, and is used to describe the progression of spermatogenesis. This description is based primarily on the changes in the acrosome and the nuclear morphology of haploid spermatids. However, such criteria cannot be applied under pathological conditions with arrested spermatid differentiation. In such settings, the changes associated with the differentiation of premeiotic germ cells must be analyzed. Here, we found that the unique bipolar motor protein, KIF11 (kinesin-5/Eg5), which functions in spindle formation during mitosis and meiosis in oocytes and early embryos, is expressed in premeiotic germ cells (spermatogonia and spermatocytes). Thus, we aimed to investigate whether KIF11 could be used to describe the progression of incomplete spermatogenesis. Interestingly, KIF11 expression was barely observed in haploid spermatids and Sertoli cells. The KIF11 staining allowed us to evaluate the progression of meiotic processes, by providing the time axis of spindle formation in both normal and spermatogenesis-arrested mutant mice. Accordingly, KIF11 has the potential to serve as an excellent marker to describe spermatogenesis, even in the absence of spermatid development. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the effect of replacing Freeze-drying (Lyophilization) low-density lipoprotein for different periods as an alternative to egg yolk, with different concentrations (6.4, 4.8, 3.2) g / 100 ml in Tris dilution, within equal periods (2h, 4h), on some Holstein bulls acrosome sperm traits, it was purified and packaged in sealed and sterile containers and kept refrigerated until by freeze-drying, low-density lipoproteins (LDLs) were filled in liquid cases in sterile cans, they were placed in a dry-freeze device. The semen was collected from 4 bulls Holstein (4-3.5 years ranging ages), using an artificial vagina with 2 ejaculation / bull / week, 1 mL of semen / bull was taken to pooled semen, to remove individual differences between bulls, the liquid was divided into the eight treatments evenly (1 ml / treatment) using the Tris dilator and within periods equal to (2h, 4h) the dilution ratio was 1:10, egg yolks were added by 20% to the control group for both periods,it was then added by lyophilization low-density lipoprotein with different concentrations of the six treatments (6.4) T1, (4.8) T2, (3.2) T3, within equal periods (2h, 4h), the effect of these additions was studied on some acrosome sperm traits (48 hours, 1 month, 2 months). The results indicate that LDL supplementation and freeze-free storage at LDL (6.4) showed a significant increase in the percentage of acrosome safety with a significant decrease in the percentage of acrosome abnormalities. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate supplementation of Melatonin to Lake 1960 chicken semen extender and semen liquid storage under 5 C to semen quality. In this study, 20 fertile Iraqi native roosters used. Semen collected from all roosters (pooled sample) in a glass tube for 3 times, 3 days per another one. Semen divided into 4 tubes and diluted 1:1 by using Lake 1960 extender with 0, 0.5, 1 and 1.5 mM/L melatonin to the extender, then stored under 5C in glass tubes for 0, 12, 24, 48, 72 and 96 hours. Sperms individual motility, viability, abnormality, and acrosomes abnormality evaluated for each treatment and time storage. The results showed that decreasing (Pd ≤ 0.0001) in sperms individual motility for all Melatonin treatments compared with control groups at 96 hours. No significant differences between Melatonin treatments and control for another storage times. Decreasing (Pd ≤ 0.05) in sperms abnormality in 1.5 mM/L Melatonin for 96-hour storage compared with other groups. Decreasing in acrosomes abnormality (Pd ≤ 0.05) in 1.5 mM/ L Melatonin for 24-and 48-hours storage. Moreover, Melatonin supplementation to semen extender improved sperms motility and decreasing sperms and acrosomes abnormality. [ABSTRACT FROM AUTHOR]

Physicochemical properties of water molecules as the main compositions of the freezing media can be affected by the electromagnetic fled. The purpose of this study was to apply extremely low repetition rate electromagnetic fields (ELEFs) to change the molecular network of water molecules existing in freezing media used for human sperm cryopreservation. First, different time periods and pulsed electromagnetic fields were used to evaluate the physiochemical properties of water. The lowest rate of cluster size, surface tension, viscosity, and density was observed for water samples exposed to 1000 Hz ELEF for 60 min (P < 0.05) that could be results in small ice crystal formation. Therefore, this treatment was selected for further evaluations in human sperm freezing because there was minimal probability of amorphous ice crystallization in this group. To assess fertilizing potential, human semen samples were subjected to ELEF (1000 Hz) water-made freezing medium and cryopreserved. The highest percentage of total motility, progressive motility, viability, membrane integrity, mitochondrial membrane potential, DNA integrity, and TAC were obtained in frozen ELEF as compared to other groups. The percentage of viable spermatozoa (Annexin V-/PI-) in frozen ELEF was significantly higher than in frozen control. The level of ROS was significantly lower in frozen ELEF when compared to frozen control. It can be concluded that the modification of physicochemical properties of water existing in cryopreservation media by ELEF is a suitable strategy to improve the outcome of cryopreservation. [ABSTRACT FROM AUTHOR]

Cilia are evolutionarily conserved hair-like structures with a wide spectrum of key biological roles, and their dysfunction has been linked to a growing class of genetic disorders, known collectively as ciliopathies. Many strides have been made towards deciphering the molecular causes for these diseases, which have in turn expanded the understanding of cilia and their functional roles. One recently-identified ciliary gene is ARL2BP, encoding the ADP-Ribosylation Factor Like 2 Binding Protein. In this study, we have identified multiple ciliopathy phenotypes associated with mutations in ARL2BP in human patients and in a mouse knockout model. Our research demonstrates that spermiogenesis is impaired, resulting in abnormally shaped heads, shortened and mis-assembled sperm tails, as well as in loss of axonemal doublets. Additional phenotypes in the mouse included enlarged ventricles of the brain and situs inversus. Mouse embryonic fibroblasts derived from knockout animals revealed delayed depolymerization of primary cilia. Our results suggest that ARL2BP is required for the structural maintenance of cilia as well as of the sperm flagellum, and that its deficiency leads to syndromic ciliopathy. [ABSTRACT FROM AUTHOR]

The high incidence of polyspermy is still an unresolved problem for the production of in vitro-produced porcine embryos. In this work, we modified the usual sperm processing sequence for in vitro fertilization (IVF), and the spermatozoa from four boars were frozen directly at a low sperm concentration of 20 × 106 sperm/mL (high pre-freezing sperm dilution group; F20), thawed and processed for IVF in three replicates. Spermatozoa from the same boars frozen at a conventional concentration (1000 × 106 sperm/mL) were used as the control group. The post-thaw sperm quality evaluation demonstrated that despite there being no differences in the percentage of motile spermatozoa between groups, the proportion of live spermatozoa with intact acrosomes was significantly higher in the F20 group than in the control. The in vitro penetration rate was also similar between groups; however, the co-incubation of oocytes with F20 sperm increased monospermy, IVF efficiency, cleavage rate and the efficiency of blastocyst formation compared with the results for oocytes co-incubated with control spermatozoa. These results indicate, for the first time, that a high pre-freezing sperm dilution increases monospermy without affecting penetration rates, thereby increasing blastocyst formation. • High pre-freezing sperm dilution improves monospermy maintaining high penetration rates and decreasing the boar variability. • High pre-freezing sperm dilution in porcine IVF increases blastocyst formation. • High pre-freezing sperm dilution increases percentages of live spermatozoa with intact acrosomes. [ABSTRACT FROM AUTHOR]

Abstract Sperm must undergo the regulated exocytosis of its dense core granule (the acrosome reaction, AR) to fertilize the egg. We have previously described that Rabs3 and 27 are organized in a RabGEF cascade within the signaling pathway elicited by exocytosis stimuli in human sperm. Here, we report the identity and the role of two molecules that link these secretory Rabs in the RabGEF cascade: Rabphilin3a and GRAB. Like Rab3 and Rab27, GRAB and Rabphilin3a are present, localize to the acrosomal region and are required for calcium-triggered exocytosis in human sperm. Sequestration of either protein with specific antibodies introduced into streptolysin O -permeabilized sperm impairs the activation of Rab3 in the acrosomal region elicited by calcium, but not that of Rab27. Biochemical and functional assays indicate that Rabphilin3a behaves as a Rab27 effector during the AR and that GRAB exhibits GEF activity toward Rab3A. Recombinant, active Rab27A pulls down Rabphilin3a and GRAB from human sperm extracts. Conversely, immobilized Rabphilin3a recruits Rab27 and GRAB; the latter promotes Rab3A activation. The enzymatic activity of GRAB toward Rab3A was also suggested by in silico and in vitro assays with purified proteins. In summary, we describe here a signaling module where Rab27A-GTP interacts with Rabphilin3a, which in turn recruits a guanine nucleotide-exchange activity toward Rab3A. This is the first description of the interaction of Rabphilin3a with a GEF. Because the machinery that drives exocytosis is highly conserved, it is tempting to hypothesize that the RabGEF cascade unveiled here might be part of the molecular mechanisms that drive exocytosis in other secretory systems. Highlights • Rabphilin3a is a Rab27 effector in human sperm. • Rabphilin3a recruits the Rab3GEF GRAB; both are required for sperm exocytosis. • Calcium requires GRAB to increase the population of cells with active Rab3 and Rap1. • Rabphilin3a and GRAB connect Rab27 to Rab3. • Rab27/Rabphilin3a/GRAB/Rab3 constitutes a signaling module in sperm exocytosis. [ABSTRACT FROM AUTHOR]

Sperm morphology and morphometry are important parameters in predicting fertility. Sperm are considered to be normal if the shape and size of the head, midpiece and tail fall within the classification for a given species. It is important to select the appropriate technique for staining the semen of a given species, because, as many authors have pointed out, some methods work well for one species but are not suitable for analysing another. The aim of the study was to assess the morphometric parameters of boar sperm following the use of different staining techniques and to verify the hypothesis that the staining technique affects the morphometric parameters of sperm. The staining method was found to significantly affect the dimensions of the boar sperm head. The semen stained by the SpermBlue technique had the closest morphometric sperm head parameters to those of the unstained sperm, so this technique, rather than the routinely used eosin and gentian complex, should be the leading technique in the evaluation of boar sperm morphometry. Silver nitrate staining reveals the structure of the sperm in the most detail; this method can be considered universal, and can be used independently or to supplement routine diagnostics. As the staining technique should interfere as little as possible with the structure of the sperm, while revealing its morphology in as much detail as possible, it is crucial to establish the natural dimensions of the unstained sperm head before determining the optimal technique and its reference values. The recommended or most commonly-used techniques are not always the best options for the staining and analysis of sperm of a given species. [ABSTRACT FROM AUTHOR]

The spermatogenesis of Porifera is still relatively poorly understood. In the past, it was accepted that all species presented a primitive-type spermatozoon, lacking special structures and acrosome. Nonetheless, a very peculiar spermatogenesis resulting in a sophisticated V-shaped spermatozoon with an acrosome was found in Poecilosclerida. This finding called into question the diversity and evolution of sperm cells in Poecilosclerida and in Porifera, as a whole. Is this sophisticated spermatozoon widespread in the group? We investigated here the ultrastructure of the spermatogenesis of three different species of Poecilosclerida (Demospongiae): Iophon proximum, I. piceum, and Tedania ignis. In all three species, spermatogenesis was synchronized within the spermatic cysts. Iophon proximum and T. ignis presented elongated sperm cells, but in I. piceum, their shape was oblong. A structure resembling an acrosome was only found in T. ignis. All three species presented modified spermatozoa, as in the majority of poecilosclerids investigated so far. Therefore, we are proposing that the elongated shape (modified type) of spermatozoon is the ancestral state based in the current phylogeny of Poecilosclerida and that the V-shaped sperm is only found in Crambeidae. [ABSTRACT FROM AUTHOR]

Sperm structure of the Xenacoelomorpha (Acoelomorpha plus Xenoturbellida) is updated in the light of new discoveries or new interpretations of existing data. Nemertodermatida and Acoela (Acoelomorpha) have introsperm with certain basic features in common, but all acoels lack acrosomes and usually have two flagella with unusual combinations of microtubules, whereas all nemertodermatids have small, simple acrosomes and a typical 9 + 2 flagellum. Xenoturbellida is currently considered as the sister taxon to Acoelomorpha. Xenoturbella bocki has an aquasperm that has almost nothing in common with the sperm of Acoelomorpha. We argue that the aquasperm ultrastructure of X. bocki has much in common with sperm of hemichordates and to some extent echinoderms, which was previously disputed. Molecular analyses have on the one hand supported a connection with deuterostomes but on the other hand have negated it, suggesting that the closest common ancestor of Xenacoelomorpha is either the Nephrozoa, Deuterostomia or Protostomia. Sperm structure is highly diverse among Xenacoelomorpha, with protostome-like traits in Acoelomorpha and deuterostome-like traits in Xenoturbella. Assuming Xenacoelomorph monophyly and ancestral introsperm in this taxon, however, suggests that the re-expression of the aquasperm form of Xenoturbella, involving some key changes in sperm morphology, is a secondarily derived state that could have occurred through "progenetic spermiogenesis" with the precocious development of round spermatids to maturity. [ABSTRACT FROM AUTHOR]

Abstract The objective of this study was to evaluate the effect of different freezing rates and thawing temperatures on the post-thaw quality of camel spermatozoa. Ten ejaculates from five male camels were frozen at five different freezing rates, achieved by placing the straws at specific heights above the surface of liquid nitrogen for different lengths of time (4 cm for 15 min; 1 cm for 15 min; 7 cm for 15 min; 7 cm for 5 min + 4 cm for 3 min; 4 cm for 5 min + 1 cm for 3 min) followed by storage in liquid nitrogen. Two thawing temperatures (37° for 30 s and 60 °C for 10 s) were subsequently tested. Post-thawing, the samples were evaluated for total and progressive motility, kinematics, membrane and acrosome integrity, and membrane functionality (hypoosmotic swelling test) at zero and 1 h post thawing. Total and progressive motility were significantly higher for the fastest freezing rate (at 1 cm) at 0 h (p < 0.01 for both), as were VCL (p < 0.01), VSL (p < 0.05) and STR (p < 0.05). Freezing at 4 cm produced the lowest values of STR compared to other treatments (p < 0.05). At 1 h, no differences in total motility were observed between freezing at 4 cm and 1 cm, both being significantly better than freezing rate 7 cm + 4 cm (p < 0.01). For progressive motility and VSL, only freezing at 1 cm was superior to the 7 cm + 4 cm combination (p < 0.001 and p < 0.05 respectively). Membrane integrity at 1 h was higher for freezing at 7 cm than at 1 cm (p < 0.01). For thawing temperatures, total motility and progressive motility at 0 h and 1 h (p < 0.001), and acrosome integrity at 1 h (p < 0.01) were higher for 60 °C thawing temperature than 37 °C. The kinematics VCL (p < 0.001), VSL and STR (p < 0.01), and VAP (p < 0.05) showed higher values for 60 °C thawing temperature than 37 °C at 0 h. After 1 h, higher values for VSL, VCL and VAP (p < 0.05) were observed for 60 °C than for 37 °C. In conclusion, a fast freezing rate would probably be beneficial for camel semen, and thawing should be conducted at 60 °C. [ABSTRACT FROM AUTHOR]

Abstract Seminal plasma (SP) plays a vital role in the maintenance of sperm function and integrity along with being involved in their communication with the female reproductive tract. Under in vitro conditions, although it is generally accepted that boar semen is better preserved when SP is diluted (extended) or removed (cryopreserved), its role during storage is not completely elucidated. In this context, the current study sought to determine the role of SP during storage of boar spermatozoa at 17 °C for 72 h. Thus, two treatments were prepared with semen from the sperm-rich fraction (SRF) of boar ejaculate previous to storage in liquid state: 1) PSP: semen directly extended in Beltsville Thawing Solution (BTS), and 2) ASP: semen first centrifuged with subsequent removal of supernatant (containing SP) followed by suspension of sperm in BTS. From this, two experiments were conducted separately in this work: 1) in vitro and 2) in vivo assays. The former aimed to evaluate how sperm capacity responds to in vitro capacitation (IVC) and whether their quality is affected by previous exposure to SP. In the latter, the objective was to understand how important these previous conditions can be for reproductive performance after artificial insemination (AI). According to our results, the previous removal of SP does not affect sperm quality and the response of these cells to IVC (P > 0.05) along with resulting in a lower percentage of acrosome damage in them [12.87 ± 0.76 (ASP) vs. 16.38 ± 0.73 (PSP)] (P < 0.05). This improved preservation of acrosome integrity in the absence of SP can explain the higher fertility rate (%) [63.27 ± 23.47 (ASP) vs. 38.57 ± 16.30 (PSP)] and number of implanted embryos at 28 days after AI (13.71 ± 4.88 (ASP) vs. 7.16 ± 4.02 (PSP)] (P < 0.05) presented by gilts inseminated with seminal doses of ASP. In conclusion, removal of SP prior to liquid storage of boar sperm for 72 h can be beneficial for their fertilizing ability. Highlights • Boar sperm stored in absence of seminal plasma was not deleterious to spermatozoa viability. • Responsiveness of boar sperm stored in absence of seminal plasma to in vitro capacitation is maintained. • Boar sperm stored in absence of seminal plasma was not deleterious to motility parameters. • Boar sperm stored in absence of seminal plasma present greater resistance to the acrosomal damage. • Better reproductive performance can be achieved in boar sperm stored without seminal plasma. [ABSTRACT FROM AUTHOR]

Abstract Boar semen cannot be immediately cryopreserved, it need be hold at 17 °C prior to cryopreservation, holding time has been used to improve cryopreserved boar semen, since holding time allows a prolonged interaction between spermatozoa and seminal plasma components. However, until now only few periods of holding time have been studied, and boar semen had been held at 17 °C for 24 h to facilitate its manufacture. Thus, this experiment aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32 h) on boar spermatozoa post-thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were extended in Beltsville Thawing Solution and storage at 17 °C. After each holding time (0, 4, 8, 12, 24, 28 and 32 h), a sample was centrifuged, and sperm pellet was diluted in an extender composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential, detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and acrosome membranes. To mitochondrial membrane potential, 32 h is needed. However, holding time was not able to control the superoxide anion amount neither membrane lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT boar semen the ideal holding time is 24 h. Highlights • Boar semen cannot be immediately cryopreserved. • The ideal holding time for boar semen is 24 h at 17 °C prior to cryopreservation. • Holding time allow a better interaction among seminal plasma molecules and spermatozoa. • Hold boar semen prior to cryopreservation increases sperm motility and membranes integrity. • Holding time was not efficiently to control oxidative damages. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effects of sperm treatment medium—TCM199 or EGTA in Tris‐HCl buffer (TBS + EGTA)―for sonication of frozen‐thawed hamster spermatozoa in terms of sperm chromosome integrity and development of hamster oocytes injected with the sperm heads (ICSI). Methods: Frozen‐thawed hamster spermatozoa were separated into heads and tails by sonication in TCM199 or TBS + EGTA. Sperm heads were injected into mouse oocytes to assess hamster sperm chromosomes. We further compared the development of hamster ICSI embryos produced by injecting sonicated sperm heads in TCM199 vs TBS + EGTA. Results: Sperm chromosome integrity was greater following sonication of frozen‐thawed hamster spermatozoa in TBS + EGTA than in TCM199 (89.7% vs 69.0%). Embryonic development was improved following hamster oocyte injection with sperm heads sonicated in TBS + EGTA compared to in TCM199 (8‐cell: 84.1% vs 65.4%; morula: 78.4% vs 43.2%; blastocyst: 42.0% vs 17.3%). Gene expression of zygotic genome activation in 2‐cell embryos was significantly higher with TBS + EGTA than with TCM199. We transferred 43 morulae/blastocysts from the TBS + EGTA group to foster mothers, and 4 (9.3%) developed into live offspring. Conclusion: These results showed that the rapid injection of hamster sperm heads separated by sonication in TBS + EGTA effectively produced more ICSI embryos during a short time. [ABSTRACT FROM AUTHOR]

Wild African Lion Panthera leo populations are decreasing due to inbreeding and reduced genetic variability. Thus, the use of assisted reproduction in the species could one day become essential. Before this is possible, however, studies need to be conducted on the basic reproductive traits of the species, especially those regarding sperm cells. This study aimed to analyze the semen of African Lions in field conditions. We included seven captive African Lions in our study. The animals were chemically restrained and electroejaculated. Twenty sperm samples were selected and analyzed for sperm motility and progressive motility, sperm motility index, and sperm morphology. In addition, the samples were analyzed for membrane and acrosome integrity (hypoosmotic swelling test and fast green/rose Bengal dyes, respectively) and assessed for cytochemical activity of the mitochondria. We found that sperm motility rate was 75.25%±2.03, progressive motility rate was 3.25%±0.10, and sperm motility index was 70.12%±1.71. We found morphologic abnormalities roughly at the expected rate with 34.61%±7.22 of the sperm cells having an intact plasma membrane and acrosome integrity of 92.27%±2.73; high mitochondrial activity was 54.26±4.88% and absence of mitochondrial activity was 2.72±0.68% in the sperm cells. These findings show that conventional tests for sperm motility and sperm morphology bring about the expected results for lions according scientific literature. Though a hypoosmotic swelling test may be performed using different concentrations, it might lead to a higher number of sperm cells with membrane damage. Fast green/rose Bengal stain and 3'3 diaminobenzidine assay, however, can be used in sperm analysis of lions in field conditions. [ABSTRACT FROM AUTHOR]

NWC is an uncharacterised protein containing three strongly conserved domains not found in any other known protein. Previously, we reported that the NWC protein is detected in cells in the germinal layer in murine testes (strain: C57BL/6), and its knockout results in no obvious phenotype. We determined the NWC expression pattern during spermatogenesis, and found this protein in spermatocytes and round spermatids, but not in epididymal sperm. Although NWC knockout males are fertile, we further characterised their reproductive potential employing non-standard mating that better simulates the natural conditions by including sperm competition. Such an approach revealed that the sperm of knockout males fail to successfully compete with control sperm. After analysing selected characteristics of the male reproductive system, we found that NWC knockout sperm had a reduced ability to fertilize cumulus-intact eggs during IVF. This is the first report describing a subtle phenotype of NWC knockout mice that could be detected under non-standard mating conditions. Our results indicate that NWC plays an important role in spermatogenesis and its deficiency results in the production of functionally impaired sperm. [ABSTRACT FROM AUTHOR]

Contents: The study was designed with three experiments to evaluate the effects of pre‐freeze supplementation of Nigella sativa oil (NSO) and thymoquinone (TQ) on total motility, progressive motility, biokinetic characteristics, acrosomal integrity and DNA integrity of cryopreserved ovine spermatozoa. Semen samples collected from three proven fertile Merino rams were diluted with a Tris‐based cryomedia containing different levels of NSO (Experiment I: 0, 10, 100 and 1,000 g/ml), TQ (Experiment II: 0, 1, 10 and 20 g/ml) and their optimum levels (Experiment III: 100 g/ml of NSO, 10 g/ml of TQ and 1 mM of α‐tocopherol and cryopreserved as pellet (200µL) and subsequently evaluated at different post‐thaw incubation periods (0, 2 and 4 hr). The results revealed that the percentage of total motility, progressive motility and biokinetic characteristics such as average path velocity, curvilinear velocity and straight‐line velocity were higher (p < 0.05) in the sperm aliquots cryopreserved with 100 g/ml NSO or 10 g/ml TQ than in the sperm aliquots cryopreserved without supplementation just after thawing and 2 hr of post‐thaw incubation. Among the supplements, NSO (100 g/ml) showed higher values of the total motility, progressive motility, biokinetic characteristics specially, average path velocity, curvilinear velocity and straight‐line velocity, acrosome integrity and DNA integrity compared with the spermatozoa frozen without supplementation. Therefore, the results suggest that NSO may be added to the cryomedium to improve the cryosurvival of ovine spermatozoa. [ABSTRACT FROM AUTHOR]

Contents: The aim of this study was to investigate the effect of varying hydrostatic pressure treatments (HP) on the boar semen quality during the modified cryopreservation. In Experiment I, combinations of pressure level (20/40/80 MPa) and duration of application (40/80/120 min) were used. Before freezing, only the magnitude but not the duration influenced the total (TM%) and progressive motilities (PM%). The 20/40 MPa levels yielded a significant (p < 0.05) improvement compared to control samples (atmospheric), but the 80 MPa was detrimental. The post‐freezing‐thawing (FT) motilities were influenced significantly by both the HP level and its duration. For TM%, the 40 MPa:120 min gave the highest post‐FT result (54.8% ± 3.3%); however, the 40 MPa:80 min (41.0% ± 3.1%) application showed the largest and significant improvement (18.4% ± 3.1%) compared to its control (22.6% ± 3.1%) and compared to the improvement (12.9% ± 3.6%) achieved by 40 MPa:120 min. For PM%, the improvement with the 40 MPa:120 min application was slightly larger than with the 40 MPa:80 min one (15.2% ± 4.2% vs. 13.8% ± 3.3%); furthermore, the difference was not significant. In Experiment II, the 40 MPa:80 min combination was tested at four different stages of the semen handling. By pressurization after dilution with the freezing extender without glycerol, significantly higher post‐FT values (TM%, intact acrosome% and head membrane%) were obtained. The two experiments demonstrated possible improvement in post‐FT semen quality achievable through the appropriate application of HP to boar semen during cryopreservation. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Each germ cell cluster in acinus is entirely covered by Sertoli cells. • Each cluster is composed of germ cells of the same developmental stage. • Clusters of different stages are randomly arranged along the acinus periphery. • The central region of the acinus is occupied by spermatozoa. • The germ cell arrangement seen in this species may be characteristic of Unionoidea. Abstract Spermatogenesis in the freshwater pearl mussel Margaritifera laevis was investigated using light and electron microscopy. The testes of M. laevis are composed of numerous acini. We observed type A spermatogonia, large cells of irregular shape, solely near the acinus basal lamina. Type A spermatogonia proliferate and become type B spermatogonia, which are also irregular in shape and form clusters of germ cells of the same developmental stage. The numerous clusters differ with respect to developmental stage and are arranged randomly along the acinus periphery. The central region of the acinus was observed to contain only mature spermatozoa. This germ cell arrangement contrasts that of other bivalvians and may be characteristic of Margaritiferidae and Unionidae. We noted that each germ cell cluster is entirely covered throughout spermatogenesis by Sertoli cells that are loosely bound together. This report is the first to describe the involvement of Sertoli cells in Unionoidea spermatogenesis. Mature spermatozoa of M. laevis are of the primitive sperm type, having a cylindrical head with a discoidal acrosome and a midpiece with five spherical mitochondria. [ABSTRACT FROM AUTHOR]

Calcium signalling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility and acrosome reactions are all mediated by increases in intracellular Ca2+. Our previous reports have shown that deficiency of MTMR14, a novel phosphoinositide phosphatase, induces a muscle disorder by disrupting Ca2+ homeostasis. Recently, we found that MTMR14 is also expressed in the testes; however, whether deficiency of MTMR14 in the testes also alters the Ca2+ concentration and impairs male fertility remains entirely unknown. In the present study, we found that MTMR14 is also expressed in the testes and mature sperm cells, suggesting that deficiency of MTMR14 might have some effect on male fertility. Both in vivo fertility and in vitro fertilization tests were then performed, and we found that MTMR14-/- male mice showed decreased fertility. A series of experiments were then arranged to test the testis and sperm parameters; we found that MTMR14 deficiency caused small size of the testes, small numbers of both total and immotile sperm, expanded membrane of sperm tail, a decreased proportion of acrosome reaction, and in contrast, an increased proportion of abnormal sperm and augmented apoptosis, etc. Further study also found that the muscle force of the vas deferens decreased significantly in KO mice. Intracellular calcium homeostasis in the testes and epididymis was impaired by MTMR14 deletion; moreover, the relative mRNA expression levels of Itpr1, Itpr2, and Ryr3 were dramatically decreased in MTMR14 KO mice. Thus, MTMR14 deletion impairs male fertility by causing decreased muscle force of the vas deferens and intracellular calcium imbalance. [ABSTRACT FROM AUTHOR]

Abstract Genome Resource Banks are keystones in the ex-situ conservation of wild species. Post-mortem (PM) collection of epididymal spermatozoa is an opportunistic and valuable source of germplasm, the time from the death of the animal limits its use. Seeking to improve germplasm preservation strategies for the chamois (Rupicapra sp.), the effect of PM time on epididymal sperm quality and freezability was studied using the Cantabrian chamois. Samples were classified according to PM collection time, up to 216 h (refrigerated), and cryopreserved (Tris-citric acid-fructose, 430 mOsm/kg, 15% egg yolk, 8% glycerol; freezing at −20 °C/min). Sperm quality was assessed after recovery and post-thawing (motility by CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). The sperm mass pH and osmolality showed a positive correlation with time. Total sperm motility dropped after 2 days PM, with progressivity and sperm velocities remained similar up to 3 days PM. Sperm freezability was acceptable, with the post-thawing HOST, motility, progressivity, VAP, VCL, VSL and BCF negatively correlating with PM time. Overall, chamois epidydimal samples were not adequate for preservation after 6 days PM. Freezability capacity could make these spermatozoa suitable for specific ART even if kept refrigerated for several days PM. [ABSTRACT FROM AUTHOR]

Highlights • Centrifugation at 500 x g for 10 min was not deleterious to spermatozoa viability. • Boar sperm cooled in absence of seminal plasma decrease of motility parameters. • Loss of plasma membrane integrity in boar sperm cooled without seminal plasma. • Boar sperm cooled in absence of seminal plasma loss integrity of acrosome. Abstract Seminal plasma (SP), the fluid that surrounds the sperm cells, is known to exert substantial influence on sperm physiology. The SP has a pivotal role in sperm function in vivo, and due to its components, it functions in an ambiguous manner in vitro , simultaneously possessing deleterious and beneficial effects. This experiment aimed to describe the differences between the presence or absence of SP from the sperm-rich fraction on some spermatozoa characteristics (kinetics, plasma and acrosome membrane integrity, lipid peroxidation and capacitation-like changes). Furthermore, this experiment focused on distinguishing the effects of SP on the variables evaluated from the effects of centrifugation during SP removal. Total and progressive sperm motility, as well as integrity of plasma and acrosome membranes, were less (P < 0.05) in the absence of SP. Membrane lipid peroxidation (P < 0.05) and sperm membrane stability (P < 0.05) did not differ among treatments. The SP from the sperm-rich fraction is important for the maintenance of adequate structural and functional characteristics of extended liquid boar semen and should be present in seminal doses throughout storage. Furthermore, the detrimental effect on the variables evaluated was caused solely by the absence of SP and not by the process of removal through centrifugation at 500 x g for 10 min. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights • Rosmarinic acid (RA) treatment decreases motility of human sperm. • RA inhibits capacitation and spontaneous acrosome reaction of human sperm. • RA reduces human sperm intracellular Ca2+ concentration ([Ca2+] i). • RA impairs human sperm [Ca2+] i by affecting KSPER. Abstract Rosmarinic acid (RA), a natural phenolic ester, is cytoprotective for male reproduction in animal models. The present study investigated the in vitro actions of RA on human sperm functions. Human sperm were exposed to 1, 10, 100, and 1000 μM RA in vitro and sperm functions were examined. The results showed that although RA did not affect human sperm viability, RA at 10–1000 μM dose-dependently reduced sperm motility, penetration ability, capacitation, and spontaneous acrosome reaction. In addition, the intracellular Ca2+ concentration ([Ca2+] i), which serve as a key regulator of sperm function, was decreased by RA (10–1000 μM) in a dose-dependent manner. Furthermore, the current of the sperm-specific potassium channel, KSPER, which is predominant for Ca2+ influx in sperm, was dose-dependently inhibited by 10–1000 μM RA. Therefore, we conclude that in vitro exposure to RA can compromise human sperm functions by decreasing sperm [Ca2+] i through the suppression of KSPER current. [ABSTRACT FROM AUTHOR]

Highlights • CBD has biphasic effects on male copulation. • CBD negatively affected the fertilization rate and the number of litters. • The antifertility properties indicate that CBD can act on the sperm acrosome reaction. Abstract Cannabidiol (CBD) is a potent substance extracted from Cannabis sativa. As it has been suggested that marijuana can affect the reproductive system, we decided to assess the effects of chronic CBD exposure on the male reproductive system in an animal model. 21-day old male Swiss mice received CBD for 34 consecutive days at doses (p.o.) of either 15 or 30 mg/kg, and a control group received sunflower oil. Body weight gain and circulating progesterone concentration did not significantly change in CBD-treated animals. In the sexual behavior analysis, the CBD 15 group presented a delay in performing the first mount and intromission, and a reduced number of mounts and ejaculations. The CBD 30 group showed a 30% reduction in fertility rate and a 23% reduction in the number of litters. Our results indicate that chronic CBD exposure promotes functional impairment of the reproductive system of male Swiss mice. [ABSTRACT FROM AUTHOR]

Summary: This work sought to address the effects of melatonin during in vitro capacitation (IVC) and progesterone‐induced acrosome exocytosis (IVAE) in boar spermatozoa. With this purpose, two different experiments were set. In the first one, IVC and IVAE were induced in the absence or presence of melatonin, which was added either at the start of IVC or upon triggering the IVAE with progesterone. Different parameters were evaluated, including intracellular levels of peroxides and superoxides, free cysteine radicals and distribution of specific lectins. While melatonin neither affected most capacitation‐associated parameters nor IVAE, it dramatically decreased sperm motility, with a maximal effect at 5 μm. This effect was accompanied by a significant increase in the percentage of agglutinated spermatozoa, which was independent from noticeable changes in the distribution of lectins. Levels of free cysteine radicals were significantly lower in melatonin treatments than in the control after 4 h of incubation in capacitating medium. The second experiment evaluated the effects of melatonin on in vitro fertilising ability of boar spermatozoa. Spermatozoa previously subjected to IVC in the presence of 1 μm melatonin and used for in vitro fertilisation exhibited less ability to bind the zona pellucida (ZP) and higher percentages of monospermy. In conclusion, melatonin affects sperm motility and the stability of nucleoprotein structure and also modulates the ability of in vitro capacitated boar spermatozoa to bind the oocyte ZP. However, such effects do not seem to be related to either its antioxidant properties or changes in the sperm glycocalix. [ABSTRACT FROM AUTHOR]

High-fat diet is associated with hypercholesterolemia and seminal alterations in White New Zealand rabbits. We have previously reported disorders in the development of the manchette-acrosome complex during spermiogenesis and decreased testicular efficiency in hypercholesterolemic rabbits. On the other hand, olive oil incorporated into the diet improves cholesterolemia and semen parameters affected in hypercholesterolemic rabbits. In this paper, we report the recovery—with the addition of olive oil to diet—from the sub-cellular mechanisms involved in the shaping of the sperm cell and testicular efficiency altered in hypercholesterolemic rabbits. Using morphological (structural, ultra-structural and immuno-fluorescence techniques) and cell biology techniques, a reorganization of the manchette and related structures was observed when olive oil was added to the high-fat diet. Specifically, actin filaments, microtubules and lipid rafts—abnormally distributed in hypercholesterolemic rabbits—were recovered with dietary olive oil supplementation. The causes of the decline in sperm count were studied in the previous report and here in more detail. These were attributed to the decrease in the efficiency index and also to the increase in the apoptotic percentage in testis from animals under the high-fat diet. Surprisingly, the addition of olive oil to the diet avoided the sub-cellular, efficiency and apoptosis changes observed in hypercholesterolemic rabbits. This paper reports the positive effects of the olive oil addition to the diet in the recovery of testicular efficiency and normal sperm shaping, mechanisms altered by hypercholesterolemia. [ABSTRACT FROM AUTHOR]

At times when rhinoceros are fiercely poached, when some rhinoceros species are closer than ever to extinction, and when the scientific community is in debate over the use of advanced cell technologies as a remaining resort it is time to simplify and improve existing assisted reproduction techniques to enhance breeding and genetic diversity in the living populations under our care. Semen cryopreservation has been performed in all captive rhinoceros species with limited degree of success. Here we tested three freezing extenders, containing different cryoprotectants and various freezing rates for the cryopreservation of rhinoceros sperm from 14 bulls. In experiment I, semen from 9 bulls was used to determine the most suitable diluent, cryoprotectant and freezing rate for the successful cryopreservation of rhinoceros sperm. In experiment II, semen from 5 bulls was used to assess whether the removal of seminal plasma could further improve post thaw sperm quality following cryopreservation with conditions identified in Experiment I. Semen was diluted with Berliner Cryomedia, ButoCrio® or INRA Freeze®, packaged in 0.5 mL straws and frozen 3, 4, and 5 cm over liquid nitrogen (LN) vapour or directly in a dryshipper. It was found that semen extended with ButoCrio® (containing glycerol and methylformamide) and frozen 3cm over LN vapour provided the best protection to rhinoceros spermatozoa during cryopreservation. When pooled over treatments, total and progressive post thaw motility was 75.3 ± 4.2% and 68.5 ± 5.7%, respectively marking a new benchmark for the cryopreservation of rhinoceros sperm. Post thaw total and progressive motility, viability and acrosome integrity of semen diluted in ButoCrio® was significantly higher than semen extended in Berliner Cryomedia or INRA Freeze®. The removal of seminal plasma did not improve post thaw sperm survival (p > 0.05). In conclusion, the cryosurvival of rhinoceros spermatozoa was significantly improved when using a mixture of glycerol and methylformamide in combination with a fast freezing rate at 3 cm. These results describe a new protocol for the improved cryosurvival of rhinoceros spermatozoa and will enable a more successful preservation of genetic diversity between males, especially in donors whose spermatozoa may already be compromised prior to or during collection. The successful reduction of glycerol concentration in favour of methylformamide as a cryoprotectant could be a novel suggestion for the improvement of cryopreservation techniques in other wildlife species. [ABSTRACT FROM AUTHOR]

Sperm samples used on fertilization strongly influence the in vitro production (IVP) rates. However, sperm traits behind this effect are not stated consistently until now. This study aimed to evaluate the isolated and combined effect of some sperm traits (MB: total motility before Percoll® gradient, MA: total motility after Percoll® gradient, AI: acrosome integrity, MI: membrane integrity, MP: mitochondrial membrane potential, and CR: chromatin resistance) on IVP rates. This is the first study focusing on the isolated effect of distinct traits. For this purpose, the experiment was divided in three steps. In first step, to study behavior of traits sperm samples (n = 63 batches) were analyzed and ranked based on each trait. In second step, samples ranked were selected from target ranks regions and allocated in groups of four to five batches, creating Higher and Lower groups, according to two different approaches. One aimed to form groups that differed to all sperm traits simultaneously (effect of combined traits). The other aimed to form groups that differed only to a single sperm trait while no differences were observed for the remaining traits (effect of each isolated trait). In third step, for each group successfully formed in step 2, sperm samples were individually and prospectively used for IVP. Cleavage, embryo development and blastocyst rates were recorded and compared between Higher and Lower of respective trait groups. Surprisingly, evaluation of isolated effects revealed that lower levels of MB, AI and MP resulted in higher embryo development and blastocyst rates (p<0.05), which was not observed on cleavage rate. We conclude that sperm traits strongly influence embryo development after in vitro fertilization (IVF), affecting the zygote competence to achieve blastocyst stage. Individually, levels of MB, AI or MP could be some of the key traits that may define IVP efficiency on current systems of embryo production. [ABSTRACT FROM AUTHOR]