CASA System Research Articles

Viability longevity and quality of epididymal sperm stored in the liquid state of European red deer (Cervus elaphus elaphus)

The aim of the study was to evaluate viability longevity and quality of liquid-stored epididymal sperm of European red deer (Cervus elaphus elaphus). Sperm samples were recovered from epididymides of 15 mature stags. Samples were diluted to 100 × 106 sperm⁄mL in modified Salomon’s extender and stored at 5 °C for 25 days. Sperm were analyzed to determine total motility (TMOT), progressive motility (PMOT) and motion variables [CASA system], acrosomes with normal apical ridges (NAR), viability and acrosomal status (FITC-PNA/PI), plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) on the first day (D1) and every other day of storage (D3-D25). Data were analyzed using a one-way analysis of variance (ANOVA). Spermatozoa remained motile until D25, whereas TMOT exceeded 60%, NAR and PMI exceeded 80%, FITC-PNA/PI exceeded 75%, and MMP approximated 70% until D11. There was a lesser (P < 0.05) value for these variables on D5 (relative to D1). Furthermore, there was a decrease in values for motility variables as duration of storage increased, excluding amplitude of lateral head displacement, on D3. The results indicate that liquid-stored epididymal sperm of European red deer are characterized as having a long viability (25 days) with a retention of sperm quality for as long as 11 days in the liquid storage state. In vitro or in vivo studies, however, are required to confirm the findings in the present study.

Mini-Percoll Technique Induces Similar Capacitation Features in Domestic Ruminant Frozen-Thawed Spermatozoa Regardless of the Presence of Heparin

Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modifications that render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium, heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PM and would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes in bull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quickly than buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similar whether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by mini-Percoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status and sperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed sperm after mini-Percoll processing followed by incubation with or without heparin supplementation.Materials, Methods &amp; Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percoll and supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes), capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and 18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P &lt; 0.05) in most of the sperm motility parameters after mini-Percoll. Conversely, ovine samples presented the highest (P &lt; 0.05) rate of acrosome-reacted cells after mini-Percoll. Heparin supplementation did not affect most of the parameters evaluated (P &gt; 0.05). In caprine and bovine species, a lower (P &lt; 0.05) rate of sperm agglutination was observed in the presence of heparin at 18 h of incubation. In the absence of heparin, ovine samples showed a higher (P &lt; 0.05) agglutination rate compared to the bovine species after long incubation period.Discussion: The present study compared sperm parameters (sperm kinematics, agglutination rate and capacitation status) of ruminant frozen-thawed sperm after mini-Percoll selection followed by in vitro incubation with or without heparin supplementation. In this study, it was observed the same rate of capacitated cells after the sperm selection (min-Percoll) between ruminant species. This indicate that the capacitation process occurs similarly between ruminant species, refuting the first hypothesis of this study. The presence of heparin did not influence the capacitation status of ruminant frozen-thawed sperm after mini-Percoll selection, it demonstrates that the second hypothesis was supported by this study making more economic and practical the use of ruminant frozen-thawed semen. The absence of heparin in the incubation medium did not harmed the capacitation status and sperm agglutination of ruminant frozen-thawed sperm. This supported the third hypothesis of the current study and indicate that the use of mini-Percoll technique regardless the presence of heparin could be a useful alternative for the preparation of ruminant frozen-thawed sperm. In conclusion, the capacitation status of ruminant frozen-thawed sperm is similar whether or not heparin is present after the mini-Percoll technique.

Sperm kinematics and morphometric subpopulations analysis with CASA systems: a review

Sperm kinematics and morphometric subpopulations analysis with CASA systems: a review. The subjective evaluation of seminal quality has given way to the use of objective assessment techniques by CASA technology (computer-assisted semen analysis). The application of principal components (PC) and clustering methods to reveal subpopulations of spermatozoa is a powerful tool to evaluate raw semen and processed cell suspensions, but not many researchers are aware of the technique. PC analysis is a multivariate statistical method that reduces the number of variables used in subsequent calculations used to describe the data. By integrating the original variables according to their coherence in a database into a new complex mathematical variable, clearly defined homogenous subpopulations of spermatozoa can be defined. Kinematic, morphometric, morphological or DNA integrity tests may apply to characterize it and understand the reproductive biology of the spermatozoon. In recent years there has been a substantive change in the conceptual paradigm regarding what an ejaculate is. It should be said that until recently it was considered that the representative population (billions) of sperm was made up of "equivalent" cells with a common goal: to be the one that could finally fertilize the oocyte. The verification that the set of spermatozoa is grouped in distinct subpopulations, according to their kinematic and morphometric characteristics, opens the way towards a more cooperative vision. Besides, we have seen that the subpopulation distribution is different among males, which seems to indicate different strategies that can be understood within another paradigm, that of sperm competition between different ejaculates. The existence of heterogeneous subpopulations of spermatozoa in the ejaculate that show kinematics and morphometric patterns is widely known but the biological meaning of these different sperm subpopulations is still not clear. Although the role of the different subpopulations remains unknown, the work should continue in that direction.

Effect of boar semen supplementation with recombinant heat shock proteins during summer

Artificial insemination (AI) in pigs is mainly performed with refrigerated boar semen. There is a marked negative seasonal effect on the quality of boar sperm, mainly due to relatively greater ambient temperatures; to counteract this thermal stress, sperm cells possess natural defensive mechanisms such as Heat Shock Proteins (HSPs) that prevent protein denaturation. Thus, the objective of this research was to improve the quality of commercial boar semen collected during the summer when ambient temperatures are greater using recombinant HSPs. For this purpose, different concentrations (0.1, 0.5 and 1 μg/ml) of recombinant heat shock proteins (HSPD1, HSPA8 or HSP86) were added to commercial boar semen and there was cooling for 48 h at 17 °C. After this storage period, sperm quality was assessed by analyzing sperm viability, mitochondrial membrane potential and plasma membrane lipid organization using flow cytometry; additionally, sperm motility was examined using a CASA system. Also, in vitro fertilization (IVF) using HSP-supplemented boar semen was performed and the quality of the embryos produced was evaluated using quantitative real-time polymerase chain reaction (qPCR) analyzing the relative abundance of mRNA transcripts for genes encoding for embryo quality-related proteins (BAX, TFAM, POLG and POG2). Sperm quality variables, blastocyst rates and the abundance of mRNA transcripts for the selected genes were not affected by the presence of recombinant HSPs at any concentration. These results indicate that the supplementation of commercial seminal doses with recombinant HSPs does not improve boar sperm quality or fertility during the summer months when ambient temperatures are greater.

Time and Dose-Dependent Effects of Viscum Album Quercus on Rabbit Spermatozoa Motility and Viability in Vitro.

The target of this study was to evaluate the effect of extract of the European mistletoe - Viscum album quercus L. on spermatozoa motility and viability in vitro. The CASA system was used to determine the spermatozoa motility parameters at different time intervals (0, 1, 2 and 3 h) and spermatozoa viability was determined in five different doses of Viscum album quercus L [10 (QA), 6.6 (QB), 3.3 (QC), 2.5 (QD) and 2 (QE) mg/ml]. Results in experimental groups detected a significant deterioration on rabbit spermatozoa after 1, 2 and 3 hours, compared to the control. The initial total spermatozoa motility showed increased value for all doses of Viscum album quercus in comparison to control. After in vitro culture a dose-dependent decrease (QA: reduction of 69.7 %, QB: reduction of 40.9 %) was found. For the progressive spermatozoa most significant decrease (86.8 % for QA vs. 48.5 % for QB) was detected compared to the control after 3 hours of culture. Spermatozoa viability (MTT test) was decreased in all experiment groups at the end of experiment, but the differences were not significant. Significant alterations of membrane integrity were found in groups with the highest Viscum album quercus concentration (QA, QB), but acrosome integrity showed no significant changes. Results suggest negative dose- and time-dependent effect of Viscum album quercus at higher doses on spermatozoa motility and viability parameters in vitro.

194 Libido and semen quality in intact and altered (short scrotum and castrated) post-pubertal male dairy sheep lambs

Abstract Short-scrotum (induced cryptorchid) rams can retain growth and performance characteristics of intact-rams while making them sterile and facilitating co-grazing with ewe-lambs. East Friesian x Lacaune male lambs left intact (n = 19; IN), rendered short-scrotum (n = 17; SS) or neutered (n = 25; WE) by 10 d of age, were used to evaluate libido and semen quality at 7-mo old. Lambs were managed as one group on a forage-based diet supplemented with a grain mixture (barley and soybean meal at 0.7 kg/d). Four CIDR-treated estrus-induced ewes were placed two/pen in adjacent 2.5x2.5 m pens for libido testing. Males were introduced individually to ewe pens for 5 min and reproductive behaviors recorded (anogenital sniffs, foreleg kicks, flehmen, false mounts, and service). Semen was collected from 6 random IN and SS lambs via artificial vaginas using estrus-induced ewes as teasers. Volume was recorded, and ejaculate extended in milk/egg yolk, packaged into straws, and stored at 5°C for analysis with a CASA system the following day. Behavior measurements were compared between types using PROC GLM. Ejaculate characteristics are presented as mean±SD due to insufficient sperm concentrations of SS ejaculates. Anogential sniffs, foreleg kicks, and mounts were similar in IN and SS, and higher (P &lt; 0.05) than in WE (7.44 and 6.34 vs. 4.76; 1.72 and 1.60 vs. 0.17; 1.78 and 2.05 vs. 0.22; respectively). Number of services were higher (P &lt; 0.001) in IN than SS (1.11 vs. 0.53), but time to service (112 sec) was not different. Ejaculate volume was similar in IN and SS (0.51 mL), but SS ejaculates were essentially devoid of sperm. Ejaculates from IN lambs had 0.69x109±0.48 x109 sperm, with 68.9±12.5% progressive motility, 81.8±6.1% viability and 87.1±6.1% morphologic normality. Ram lambs rendered short-scrotum by 10 d of age had reproductive behavior similar to intact post-pubertal rams, but ejaculate characteristics showed them to be sterile

Evaluation of the Spermate Quality of Cattle Players Through the use of Conventional Seminal Evaluation Systems and Casa System

Evaluation of the Spermate Quality of Cattle Players Through the use of Conventional Seminal Evaluation Systems and Casa System

Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage

Study questionDoes cryoprotection of spermatozoa using a vitrification protocol with improved cryoprotective agents and a novel device for large storage lead to better outcomes than conventional slow freezing?Summary answerVitrification of human sperm using sucrose and dextran-based cryoprotectant (CPA4) with a new vitrification device resulted in significantly better sperm motility and progressive motility and improved DNA integrity with lower DNA fragmentation compared with conventional slow freezing.What is known alreadyA major limitation to clinical implementation of vitrification is the right balance between the volume of spermatozoa suspension cryopreserved and a standardised use of CPAs for survival of spermatozoa.Study design, size, durationThis was a control versus current clinical practice study using 30 fresh human semen samples to carry out the different cryoprotectant analyses followed by a further 23 semen samples to test the novel vitrification protocol.Participants/materials, setting, methodsAll human specimens fulfilled the following criteria: > 5 million spermatozoa/mL, > 20% total motility, ≥ 1.8 mL in volume, with all participants falling within the age range of 25–45 inclusively. The concentration, progressive motility, non-progressive motility, immotility, and various morphokinetic variables including DAP, DCL, DSL, LIN, and STR were then determined using the IVOS II™ Clinical CASA system (Hamilton Thorne, Beverly, MA, USA) on the basis of the 5th Edition of WHO Laboratory Manual for the Examination and Processing of Human Semen.Main results and the role of chanceAmong the 6 cryopreservation methods in this study, vitrification with the funnel-shaped device using CPA4 best preserves the 13 sperm parameters evaluated by CASA system. Conventional slow freezing and vitrification with the device using seminal plasma also protects sperm quality, but the overall motilities are statistically lower in comparison with the novel vitrification approach with cryoprotective media using the device. DNA fragmentation significantly increased after cryopreservation through the method of conventional slow freezing (p = 0.07). There was no significant difference in DNA fragmentation between fresh control and vitrification (p = 1.000).Limitations, reasons for cautionExtensive training is required to minimise the human error in using the vitrification device to perform cryopreservation. Each operator can only handle one sample at a time with device vitrification, whereas several samples can be processed without the need for special training with conventional slow freezing.Wider implications of the findingsThe presented study shows that a new vitrification method could improve survival sperm rate. Human sperm vitrification using our novel protocol gives higher motility and progression and lower percentage of DNA fragmentation than conventional slow freezing. Our findings indicate that this method could supersede the current clinical practice in particular for patients with oligospermia as it reduces osmotic damage, time, and cost.

Monocyte-derived extracellular trap (MET) formation induces aggregation and affects motility of human spermatozoa in vitro

ABSTRACTThe presence of bacteria and/or leukocytes can alter semen quality resulting in low sperm quality and infertility. Inflammation or infection increases the numbers of PMN or macrophages/monocytes in male genital tract. Release of extracellular traps (ETs) by leukocytes has been recognized as a novel mechanism of early host innate immunity, in response to invasive pathogens. This is the first work that evaluated the mechanism of triggered ETs in monocytes co-incubated with spermatozoa or bacteria and the effect on sperm function. Selected spermatozoa and human monocytes isolated from peripheral blood were obtained by healthy donors. Two experimental models were developed, one aseptic (non-infectious) incubating spermatozoa and monocytes, and septic models (infectious) incubating spermatozoa with monocytes and uropathogenic Escherichia coli (E. coli). ETs of monocytes (METs) (DNA, global histone and citrullinated histones) were visualized by scanning electron microscopy (SEM) and immunofluorescence analyses. Progressive motility was performed at 0, 10, 30, 60, and 180 min after co-incubation with CASA system. SEM- and immunofluorescence-analyses revealed human spermatozoa alone or in the presence of E. coli as strong inducers METs. In aseptic model, the motility decreased to 65.2 ± 3.5% at 10 min of incubation and 29.3 ± 3.3% at 30 min (p < 0.001). In septic model, motility decreased to 44.5 ± 5.9% (10 min) and 12.7 ± 2.2% (30 min) (p < 0.001). MET-derived small spermatozoa aggregations were observed in both models. METs might physically block spermatozoa and decrease motility after a brief contact. This may impair male fertility, especially in patients with genital tract infections or chronic inflammation.Abbreviations: PMN: polymorphonuclear; ETs: extracellular traps; E. coli: Escherichia coli; METs: ETs of monocytes; SEM: scanning electron microscopy; NE: neutrophil elastase; MPO: myeloperoxidase; MAGI: male accessory gland infection; PBMC: peripheral blood mononuclear cells; RT: room temperature; CFU: colony forming units; CASA: computer-aided sperm analysis; H4Cit3: histone H4 citrullinated 3

Improvement of Sperm Motility Within One Month Under Selenium and Vitamin E Supplementation in Four Infertile Dogs with Low Selenium Status.

IntroductionSignificant improvement of sperm motility within one month effected by oral supplementation of selenium and vitamin E was described in four infertile male dogs which failed to conceive in their last three matings with different bitches.Material and MethodsThe dogs (a Golden Retriever, an English Cocker Spaniel, and two Tibetan Mastiffs) were supplemented daily with selenium (Se) (0.6 mg/kg organic Se yeast) and vitamin E (vit. E) (5 mg/kg) per os for 60 days. Semen was collected on days 0, 30, 60, and 90. The sperm concentration and motility parameters were evaluated by the CASA system, sperm morphology was explored by Diff-Quick staining, and live and dead spermatozoa were differentiated by eosin/nigrosin staining. The concentrations of Se and vit. E were measured in peripheral blood serum on semen collection days.ResultsBefore administration, the concentrations of Se in blood plasma were low (86.0–165.0 μg/L). After 30 days of treatment there was an observable improvement in total and progressive sperm motility and kinematic parameters (VAP, VSK, VCL, ALH, BCF, and RAPID). The percentages of live and normal morphology sperm cells were also higher. There was also an observable increase in Se and vitamin E concentrations in blood serum. Bitches were successfully mated and delivered four to six puppies.ConclusionSupplementation with Se and vit. E improved rapid sperm motility and restored fertility in infertile dogs with low Se status.

Influence of bovine follicular fluid on thawed bovine spermatozoa - assessment by CASA system and flow cytometry.

The aim of this study was to analyze the effect of bovine follicular fluid on the survival, morphology and kinetic parameters of bovine thawed spermatozoa under laboratory conditions. The semen from 5 bulls of proven fertility was incubated in follicular and physiological fluid for 8 hours. During this time assessment using the CASA system was performed. At the beginning and the end of incubation process evaluation by flow cytometry was conducted. The results of the sperm motility assessment showed a significant decrease in the analyzed parameters both in the follicular and physiological fluid. A significant reduction in all parameters characterizing movement properties in the semen incubated in the follicular fluid was found. In the physiological fluid, a similar trend was demonstrated only for the following properties: VAP, VSL, VCL, ALH, BCF. A significant difference was found for both fluids in: VCL (p=0.026), ALH (p=0.038) and LIN (p⟨0.001) at the beginning of incubation. The results of the plasma membrane integrity assessment showed a statistically significant increase in the percentage of dying sperm at the 8th hour of the incubation in the follicular fluid. In the case of semen incubation in physiological fluid, a statistically significant decrease in the percentage of live non-damaged cells was found with a simultaneous increase in the subpopulation of undamaged dead cells. Follicular fluid rapidly accelerates the capacitation process. The results of flow cytometry support the hypothesis concerning the ability of follicular fluid to prolong sperm survival.

A new Genetic Algorithm based fusion scheme in monaural CASA system to improve the performance of the speech

This research work proposes a new Genetic Algorithm (GA) based fusion scheme to effectively fuse the Time–Frequency (T–F) binary mask of voiced and unvoiced speech. The perceptual cues such as correlogram, cross-correlogram and pitch are commonly used to obtain the T–F binary mask of voiced speech. Recently, researchers use speech onset and offset to segment the unvoiced speech from the noisy speech mixture. Most of the research work which uses speech onset and offset to represent the unvoiced speech, combine the segments of unvoiced speech with the segments of voiced speech to obtain the T–F binary mask. This research work effectively fuses the T–F binary mask of voiced and unvoiced speech, instead of combining the segments of voiced and unvoiced speech using a Genetic Algorithm (GA). Moreover, a new method is proposed in this research work to obtain a T–F binary mask from the segments of unvoiced speech. The performance of the proposed GA based fusion scheme is evaluated using measures such as quality and intelligibility. The experimental results show that the proposed system enhances the speech quality by increasing the SNR with an average value of 10.74 dB and decreases the noise residue with an average value of 26.15% when compared with noisy speech mixture and enhances the speech intelligibility by increasing the CSII, NCM and STOI with an average value of 0.22, 0.20 and 0.17 as compared with the conventional speech segregation systems.

Apoptotic-like changes in epididymal spermatozoa of soft-shelled turtles, Pelodiscus sinensis, during long-term storage at 4 ºC

Apoptosis is a physiological phenomenon that has been recognized as a cause of sperm death during cryopreservation in endothermic mammals. There is, however, no data on its role in sperm death during cooled storage in ectothermic animals. In this study, spermatozoa from the epididymis of soft-shelled turtle were investigated to identify the mechanism of spermatozoa apoptotic-like changes during storage at 4 °C. In this study, there was survival of spermatozoa for more than 40 Days when stored at 4 °C. During cooled storage, sperm kinematics was evaluated using CASA system. Values for all sperm motility variables decreased during the period of storage; while for velocity curvilinear (VCL) there was a further decrease after 20 Days of storage. Results from flow cytometry analysis indicated that there was a significant increase in the percentage of apoptotic spermatozoa, but there was no change in the percentage of necrosis. Furthermore, the concentration of cellular ROS increased after 20 Days of storage at 4 °C. The results using JC-1 staining indicated there was a decrease in MMP of spermatozoa as the duration of storage at 4 °C increased. Nuclear fragmentation of spermatozoa was observed using TEM on Day 30 of storage. There were large amounts of pro-apoptotic cytochrome c (Cytc) and cleaved caspase-9/3 proteins detected using western blot analysis after 30 days of spermatozoa storage at 4 °C. These findings indicate ROS generation induces mitochondria damage after 20 days of storage at 4 °C, which can induce spermatozoa apoptotic-like changes during storage of soft-shelled turtle spermatozoa.

Effect of Age and Frequency of Collection on Quality of Jersey Bulls Semen at National Livestock Breeding Center (NLBC), Nepal

Objective: To determine the effect of age and frequency of collection on quality parameters of Jersey bull semen at National Livestock Breeding Center (NLBC), Pokhara.&#x0D; Materials and Methodology: Nine Jersey bulls were selected randomly from NLBC. To obtain the effect of age, six bulls of three different age interval (3-4, 5-7 and 8-9 years) were selected randomly. Remaining three bulls were used to determine the effect of frequency of collection on semen quality. Studies were conducted for three months (Nov/Dec to Dec/Jan, 2016). Semen quality was analyzed using CASA system and SMILE software. ANOVA and paired t- tests were applied to find out level of significance. All the statistical analyses were done using SPSS 17.0&#x0D; Results: Sperm concentration, post- thaw motility, live sperm, and abnormal sperm were significantly affected by the age of the bulls. Semen quality was found superior in the bulls belonging to 5-7 years of age intervals. Significantly higher value for semen volume, sperm concentration and post- thaw motility was obtained on second collection (P&lt;0.05). However, volume initial motility, pre-filling motility, live sperm and abnormal sperm percentage did not differ significantly among different age intervals and between the frequencies of collection.&#x0D; Conclusion: Different quality parameters of semen like volume, concentration, initial motility, pre-filling motility, post thaw motility, live and normal spermatozoa percentages were higher in 5-7 years old bulls and in second collection of semen.&#x0D; Int. J. Appl. Sci. Biotechnol. Vol 7(1): 88-95

Artificial seminal plasma improves motility and fertilisation capacity of common carp Cyprinus carpio L. sperm during one hour of storage

The quality of fish sperm is characterized by relatively high individual variation. Moreover, under artificial conditions, the collected sperm loses its motility and viability very rapidly, which may decrease fertilisation success. Therefore, techniques able to secure and maintain a high biological value of collected fish sperm are needed in hatchery practice. Herein, we used previously composed artificial seminal plasma (ASP) containing 2 mM CaCl2, 1 mM Mg2SO4, 20 mM Tris, 110 mM NaCl and 40 mM KCl (pH 7.5 and 310 mOsm kg−1) to dilute common carp sperm samples for 1 h storage prior to fertilisation. Sperm was collected from mature males and, after checking its motility (MOT) using the CASA system, was divided into high (range 55–65%) and low (range 5–10%) quality. Samples of both sperm qualities (high/low) were diluted tenfold (1: 9; sperm: extender) in ASP and stored for 24 h at 10 °C. Motility (MOT, PRG) and velocity (VCL, VSL) of low- and high-quality sperm diluted in ASP increased after 1 h of storage compared to undiluted sperm. Moreover, the fertilisation capacity of low-quality sperm (previously diluted and stored for 1 h in ASP) increased three times compared to undiluted sperm of the same quality. Prolonging the time of sperm storage in ASP to 24 h results in the further increase of MOT, PRG and VCL in the low quality samples and PRG in the high quality samples. The results of the presented study confirm the possibility of common carp sperm revitalisation using ASP. The utilisation of such sperm in hatchery practice is possible since the fertilisation capacity of sperm, previously diluted and stored for 1 h in ASP, was over 90% regardless of the initial sperm quality (high/low).

Identification of proteoforms of albumin and kallikrein in stallion seminal plasma and their correlations with sperm motility.

The aim of this study was to identify the proteoforms of albumin and kallikrein in stallion seminal plasma (SP), and to determine their correlations with sperm motility parameters. The experimental material consisted of ejaculates from 8 stallions, which were collected during the breeding and non-breeding seasons (BS and NBS, respectively). SP proteins were identified by 2-D PAGE and mass spectrometry (MALDI TOT/TOF MS). Sperm motility parameters were analyzed using the CASA system. Protein expression (integrated optical density-IOD) of albumin proteoforms 1 (ALB 1) and 2 (ALB 2) and kallikrein proteoforms 1 (KAL 1) and 2 (KAL 2) was correlated (p⟨0.05) with sperm motility parameters (total motility and progressive motility) during the BS. No significant correlations were found between the expression of albumin or kallikrein and sperm motility parameters during the NBS. The presence of correlations between the expression of ALB 1, ALB 2, KAL 1, KAL 2 and selected sperm motility parameters could suggest that the analyzed components of the SP belong to the group of fertility-associated proteins (FAPs).

Reproduction of endangered river lamprey (Lampetra fluviatilis) in controlled conditions

The research reported focuses on reproduction of the river lamprey Lampetra fluviatilis,(Linnaeus, 1758) in controlled conditions. There was specific emphasis on fish harvesting dates (autumn and spring), holding conditions and reproduction in a controlled environment. Attempts were also made to synchronize the time of ovulation among river lampreys, egg and sperm collections. Hormonal stimulation was conducted using carp pituitary homogenate (CPH) at a total dose of 4 mg/kg which allowed for shortening of the egg-laying period from 2 to 3 weeks to a few days while sustaining embryo survival rates and larvae quality. River lamprey males were found to not require hormonal treatment to yield good-quality sperm, as measured using the CASA system. River lamprey broodstocks adapted well to different manipulations in hatchery conditions when harvested in the autumn and spring. The results of the present study may be used to restore endangered natural populations of the river lamprey (egg and sperm collection, fertilization or gamete preservation) because ovulation and spermiation synchronization is very difficult to achieve without hormonal treatment in controlled conditions.

Automatic Human Sperm Concentrartion in microscopic videos

&#x0D; Background: Human sperm cell counting analysis is of significant interest to biologists studying sperm function and to medical practitioners evaluating male infertility. Currently the analysis of this assessment is done manually by looking at the sperm samples through a phase-contrast microscope using expert knowledge to do a subjective judgement of the quality.&#x0D; Aims: to eliminate the subjective and error prone of the manual semen analysis and to avoid inter and intra-laboratory inconsistencies in semen analysis test results&#x0D; Methods: In this paper we introduce a technique for human sperm concentration. Its principle is based on the execution of three steps: The first step in unavoidable. It concerns the pretreatment of the human sperm microscopic videos which consists of a conversion of the RGB color space into the YCbCr space, the “Gaussian filtering” and the “discrete wavelet filtering”. The second step is devoted to the segmentation of the image into two classes: spermatozoas and the background. To achieve this, we used an edge detection technique “Sobel Contour detector”. The third step is to separate true sperm from false ones. It uses a machine learning technique of type decision trees that consist on two classes classification based on invariant characteristics that are the dimensions of the bounding ellipse of the spermatozoid head as well as its surface.&#x0D; Results: To test the robustness of our system, we compared our results with those performed manually by andrologists. After results analysis, we can conclude that our system brings a real improvement of precision as well as treatment time which make it might be useful for groups who intend to design new CASA systems.&#x0D; Conclusion: In this study, we designed and implemented a system for automatic concentration assessment based on machine learning method and image processing techniques.

Determination of cat sperm concentration using the CASA system

This study aimed to standardize the CASA system for sperm concentration of cat epididymal samples, using a Neubauer chamber as the gold standard. Epididymal spermatozoa were collected from 44 adult cats. The sperm concentration was evaluated in the Neubauer chamber and by the CASA system at a dilution of 1:200. After assays, the ejaculates were separated into four groups according to the sperm cell count in the CASA system; A (≤ 100), B (101 to 400), C (401 to 800), and D (˃ 800). The statistical analysis measured the agreement between techniques using the Bland-Altman test. A 95% confidence interval was adopted and the statistical program MedCalc 18.2.1 was used. Groups A, B, and D presented disagreement between the CASA system and the Neubauer chamber. Only group C (401 to 800 cells per field) demonstrated agreement between methods. It can be concluded that evaluation of spermatic concentration of domestic felines by the CASA system is reliable when the reading is performed within the range of 401 to 800 cells per field, and could represent a viable alternative to the Neubauer chamber.

Effect of Age and Frequency of Collection in Quality of Jersey Bulls Semen at National Livestock Breeding Center, Nepal

The present study was undertaken to determine the effect of age and frequency of collection on quality parameters of Jersey bull semen at National Livestock Breeding Center (NLBC), Pokhara, Kaski district. Nine Jersey bulls were selected randomly from NLBC. To obtain the effect of age, six bulls of three different age interval (3- 4, 5-7 and 8-9 years) were selected randomly. Remaining three bulls were used to determine the effect of frequency of collection on semen quality. Studies were conducted for three months (Nov/Dec to Dec/Jan 2017). Semen quality was analyzed using CASA system and SMILE software. ANOVA and paired t- tests were applied to find out level of significance. All the statistical analyses were done using SPSS 17.0. Sperm concentration (0.551 ±0.004 to 1.084 ± 0.014x109), post- thaw motility (42.44±0.11 to 51.72±0.03 %), live sperm (45.62 ± 0.13 to 51.23±0.15 %), and abnormal sperm (11.57±0.12 to 19.59±0.24%) were significantly affected by the age of the bulls. Semen quality was found superior in the bulls belonging to 5-7 years of age intervals. On the other hand, volume (3.98±0.09 to 6.72±0.06 ml), initial motility (72.68±0.02 to 78.64±0.15 %) and pre-filling motility (71.34±0.17 to 77.62±0.02 %) did not differ significantly among different age intervals. Significantly, higher value for semen volume (3.98±0.12 to 6.02±0.14 ml), sperm concentration (0.572±0.119 to 1.095±0.015) and post- thaw motility (40.34±0.21 to 48.54±0.06% was obtained on second collection. However, initial motility (70.58±0.06 to 75.64±0.13 %), prefilling motility (68.11±0.12 to 71.62±0.02 %), live sperm (53.27±0.54 to 56.17±0.02 %) and abnormal sperm (12.31±0.12 to 14.3±0.47 %) did not differ significantly between the frequencies of collection. Different quality parameters of semen like volume, concentration, initial motility, pre-filling motility, post thaw motility, live and normal spermatozoa percentage were higher in 5-7 years old bulls and in second collection of semen.