Semen Evaluation: Methodological Advancements In Sperm .
Open AccessAnim BiosciVol. 34, No. 8:1253-1270 August 2021https://doi.org/10.5713/ab.21.0072pISSN 2765-0189 eISSN 2765-0235Semen evaluation: methodological advancements in spermquality-specific fertility assessment — A reviewBereket Molla Tanga1,2,a, Ahmad Yar Qamar1,3,a, Sanan Raza3,4, Seonggyu Bang1,Xun Fang1, Kiyoung Yoon5, and Jongki Cho1,** Corresponding Author: Jongki ChoTel: 82-42-821-6788, Fax: 82-42-821-8903,E-mail: cjki@cnu.ac.krCollege of Veterinary Medicine, ChungnamNational University, Daejeon 34134, Korea2 Faculty of Veterinary Medicine, HawassaUniversity, 05, Hawassa, Ethiopia3 Department of Clinical Sciences, Collegeof Veterinary and Animal Sciences, Jhang35200, Sub-campus University of Veterinaryand Animal Sciences, Lahore 54000,Pakistan4 Department of Reproduction and ArtificialInsemination, Faculty of Veterinary Medicine,Aydin Adnan Menderes University, Aydin09016, Turkey5 Department of Companion Animal, ShinguCollege, Seongnam 13174, Korea1 These authors contributed equally to thiswork.aORCIDBereket Molla Tangahttps://orcid.org/0000-0001-9901-5875Ahmad Yar Qamarhttps://orcid.org/0000-0002-0366-3913Sanan Razahttps://orcid.org/0000-0003-1819-1522Seonggyu Banghttps://orcid.org/0000-0002-2513-4887Xun Fanghttps://orcid.org/0000-0001-8714-0679Kiyoung Yoonhttps://orcid.org/0000-0003-4891-2247Jongki Chohttps://orcid.org/0000-0002-8431-0457Submitted Feb 16, 2021; Revised Mar 25, 2021;Accepted Mar 30, 2021www.animbiosci.orgAbstract: Assessment of male fertility is based on the evaluation of sperm. Semen evaluationmeasures various sperm quality parameters as fertility indicators. However, semen evaluationhas limitations, and it requires the advancement and application of strict quality controlmethods to interpret the results. This article reviews the recent advances in evaluating varioussperm-specific quality characteristics and methodologies, with the help of different assaysto assess sperm-fertility status. Sperm evaluation methods that include conventional micro scopic methods, computer-assisted sperm analyzers (CASA), and flow cytometric analysis,provide precise information related to sperm morphology and function. Moreover, profilingfertility-related biomarkers in sperm or seminal plasma can be helpful in predicting fertility.Identification of different sperm proteins and diagnosis of DNA damage has positivelycontributed to the existing pool of knowledge about sperm physiology and molecularanomalies associated with different infertility issues in males. Advances in methods andsperm-specific evaluation has subsequently resulted in a better understanding of spermbiology that has improved the diagnosis and clinical management of male factor infer tility. Accurate sperm evaluation is of paramount importance in the application of artificialinsemination and assisted reproductive technology. However, no single test can preciselydetermine fertility; the selection of an appropriate test or a set of tests and parameters isrequired to accurately determine the fertility of specific animal species. Therefore, a needto further calibrate the CASA and advance the gene expression tests is recommended forfaster and field-level applications.Keywords: Biomarkers; Fertility; Sperm Biology; Sperm Evaluation; Computer-assistedSperm Analyzers (CASA)INTRODUCTIONFertility defines the rate of reproduction and ability to propagate generations, though it isdeclining globally for the last two centuries [1]. Fertility rate measures the number of offspring that can be produced during the life span, and fecundity measures the biologicalpotential of the number of offspring that can be obtained during a lifetime [2]. In eithercase, it is determined by the proper functioning of the reproductive system, its anatomyand physiology of organs, glands, ducts, and testicles/ovaries. In male animals, fertilitycan be evaluated from the number of sperm produced with the capability to fertilize theoocyte both in in-vivo and in-vitro and is measured with the help of semen analysis, eventhough it has limitations [3]. Semen analysis is advanced, but the reliability and validity offertility determination in humans [4] and various species of animals [5] are widely controversial in the literature.Semen primarily consists of two main components: the sperm and seminal fluids; spermCopyright 2021 by Animal BioscienceThis is an open-access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution,and reproduction in any medium, provided the original work is properly cited.1253
Tanga et al (2021) Anim Biosci 34:1253-1270are produced in the seminiferous tubules of the testicles, andthe seminal fluids, in the accessory sex glands and excurrentducts. Both of these components are examined in semenanalysis in terms of sperm count and semen volume [6].Semen analysis provides information about normal operations of the testicular machinery for the production of sperm,and the fluid part. Furthermore, it evaluates the sperm toassess fertility [7] or the success of surgical procedures suchas vasectomy [8]. Semen analysis includes examining thephysical characteristics of semen (color, odor, pH, viscosity,and liquefaction), volume, concentration, morphology, spermmotility and progression, conducted repeatedly at differentintervals [9]. In domestic animals, fertility determination isvery important, particularly in male animals used for breeding purposes and artificial insemination (AI). The infertilityissues associated with sperm can adversely affect the breeding outcomes of large herds of animals [10]. Therefore, theassessment of sperm fertility in breeding bulls is of paramount importance [11-13].For a long time, semen evaluation was believed to be thesingle most important laboratory test for assessing malefertility [14] however, it still remains complex and difficultto standardize. Several confounders have made the goal ofone-test evaluation quite difficult to accomplish, such as theinability to strictly control quality to obtain meaningful results, variability among laboratories, and inability to complywith standard procedures to accomplish correct interpretations from results [15]. The structural evaluation of sperm isbased on its appearance, morphology, concentration, plasmamembrane integrity, and chromatin integrity, whereas functional evaluation is based on its motility, capacitation, andacrosomal reaction [16]. Advances in technologies enablethe assessment of sperm structure and function, such asplasma membrane and genomic constituents, and enablesbetter determination of sperm fertility potential.The structure of sperm is peculiar, and functions to transfer the male gametes and required genetic information [17],equipped with a strong flagellum to propel but devoid ofcytoplasmic organelles like ribosomes, Golgi apparatus orendoplasmic reticulum [18]. However, sperm contain moreof mitochondria, placed strategically, bases of energy sourcefor motility [19]. Structurally, even if consisted of single cellmembrane, it has two morphologically and functionallydistinct, tail to motion and head containing the genetic material [20]. Different from other cells, DNA is tightly packed,and its integrity is basis for attaining the required fertility[21]. The head has anterior end of the nuclear envelope, referred as acrosome, contains hydrolytic enzymes that helpthe sperm to penetrate the egg's outer coat by an acrosomereaction [22].In bulls, semen quantity, quality, and/or health status areresponsible for a significant percentage of reproductive fail1254www.animbiosci.orgure in cattle production [23]. As AI is widely used in cattle,semen evaluation plays an imperative role not only in thesuccessful establishment of pregnancy but also in facilitatinggenetic improvement [24], attaining breeding and milk, andmeeting production objectives [25]. In bulls, factors including age, breed, time and interval of collection, and season ofthe year affect the semen quality [26]. Computer-assistedsperm analyzers (CASA) is believed to be a better methodfor assessing the quality of fresh semen, not only as a fastermeasure but also in terms of the ability to measure multipledimensions of sperm fertility, precisely and accurately [27,28].The criteria for selecting bovine semen samples for preservation or breeding purposes rely on concentration (sperm/mL),initial motility (%), and total normal morphology of sperm(%), but the recommended criteria and points vary [13,25].There is no doubt that more stringent criteria are requiredfor the optimal evaluation of semen.In domestic animals, fertility indicators are affected byseveral factors, including the host environment and nutritional factors; however, in this review, we have focused solelyon semen evaluation methods. Semen evaluation can be conducted at different levels, that is semen and sperm, or byassessing the mechanical, physical, or functional characteristics of sperm [29], which directly or indirectly evaluates thesperm-specific quality in terms of attaining fertility. For theconvenience of this review, we start with the fertility-relatedparameters of sperm and subsequently review the differentmethods and their advancements. Moreover, we describethe measurement values and advantages and disadvantagesof specific methods in various species of domestic animals.Semen quality varies depending on animal characteristics,countries, and sometimes farms [30,31].This article reviews the developments in current semenanalysis methods and the knowledge obtained from determining the fertility of sperm. Therefore, we aimed to reviewthe progress in semen evaluation techniques and to makeconclusive remarks regarding recent advances in semen evaluation of domestic animals. This review focuses on the reliabilityand limitations of semen analysis for the diagnosis of fertility.SPERM CONCENTRATION ANDMORPHOLOGYDetermination of sperm concentration, together with theassessment of motility and morphology, is an importantmethod to determine fertility. Sperm concentration per mLof semen can be determined by counting the sperm in thechamber of a hemocytometer, a microcell, or a photometer.However, there are possibilities of underestimation and overestimation of sperm numbers depending on the types ofproducts used [32]. The calibration of devices to measureconcentration is critical to ensure accurate sperm number
Tanga et al (2021) Anim Biosci 34:1253-1270per dose and produce maximum doses per ejaculate. In addition to these methods, sperm concentration can also bedetermined with the help of CASA [33], flow cytometer [34],and NucleoCounter SP-100 [35]. CASA can also be used forinstant quantification of sperm concentration and motility.The NucleoCounter SP-100 can assess sperm concentrationand membrane integrity. NucleoCounter SP-100 is more efficient than the hemocytometer because it is quicker, simpler,objective, and precise. Moreover, it is cost-effective and moreuser-friendly compared to flow cytometry [35].A study comparing various methods used for the determination of sperm concentration revealed that flow cytometrywas the most precise method [36]. However, before performing flow cytometry, there is a requirement for preliminaryassessment of sperm count using a different method to ensure proper semen dilution (close to 250,000 sperms/mL).The spectrophotometer was found to be the second-best option for the precise determination of sperm concentration.However, it might not be an appropriate method for the assessment of sperm samples with low concentrations andvolume [36]. Standardizing a laboratory procedure for assessing sperm concentration is affected by factors, whichinclude animal species, the sample size needed, operationalfrequency, the number of samples assessed per day, and theoperational cost.Morphometric characteristics of sperm are one of the mostimportant indicators of fertility. It is universally accepted thatsperm with normal morphology have a significant effect onfertility both in vivo and in vitro, and it is an integral part ofsperm functional test [37-40]. Abnormal types are categorizedaccording to the sperm morphology, including defectivehead, neck, spacer, and default queue [41]. Different fixationand smear preparation methods are adopted for the assessment of sperm morphology. However, no optimal methodhas been standardized for any particular animal species. Highvariability exists within and between laboratories regardingthe accurate assessment of sperm morphology. The computerized analysis of sperm morphology is known as automatedsperm morphometry analysis (ASMA). ASMA enables morphological assessments of live sperm [42]. This system canefficiently classify normal and abnormal sperm by overcoming technical variations. Moreover, it provides objectiveestimation, with improved accuracy and precision of spermmorphology assay. Sperm morphology can be better elaborated if staining is applied.SPERM MOTILITYThe advancements in sperm motility assessment includethe use of the light microscope and phase-contrast microscope, using 20 and 40 objectives, which are reported toyield substantially good results [43]. The microscope shouldbe equipped with a stage warmer that can be adjusted to37 C and magnification levels should allow clear visualization of the sperm samples [44]. It is advised to avoid the useof a light microscope because of clear visualization issues,as immotile sperm are difficult to identify, especially at lowmagnification [44]. The inability to identify immotile spermmay result in false high-motility values.Motility by phase-contrast microscopyPhase-contrast microscope is generally used to determinemass motility or progressive sperm motility [45]. Mass motility is a very important parameter for assessing sperm fertilityin humans and domestic animals [46-50]. Based on the motility pattern, each sperm is categorized either as possessingprogressive motility (PR) or non-progressive motility [51].While reporting sperm motility, it is necessary to considertotal motility (PR NP) or progressive motility. In terms offertility, only the percentage of progressively motile spermwas found to be associated with pregnancy rates. Thus, differentiating PR and NP is important [52].PR is usually expressed as a percentage of sperm motility,which is suggestive of sperm fertility, indicating proper spermatogenesis and maturation of sperm during epididymaltransit [53]. In humans, the PR of sperm is mainly affectedby thiols of flagellar proteins such as outer dense fiber protein 1, which are oxidized to form disulfides during epididymaltransit and the sperm attains motility [53]. In animals, withexternal fertilization, usually, the exposure of sperm to thefertilization medium results in hypotonic shock that inducesactivation of motile function [54,55]. In mammals, cAMPsignaling pathways and adenylyl cyclase are associated withprogressive sperm motility [56,57]. The PR of human spermis associated with DNA damage and fragmentation, which isvital for successful fertilization [58].Various factors can affect the results of phase-contrast microscopy, such as, magnification, working conditions (heatedstage, ambient temperature), personal experience, and environmental factors [44,59]. Some authors have describedspecific sperm motility characteristics such as swirling oscillation in warm slide microscopy to measure the massmotility to predict fertility [48].Motility by computer-assisted sperm analysisThe CASA is the first major system to analyze motion characteristics of sperm. It provides instant quantification of spermmotility and concentration. CASA is relatively better thanphase-contrast microscopy as it can generate accurate andhighly reproducible data of different kinetic parameters ofsperm [60,61]. Furthermore, CASA largely reduces the subjectivity by overcoming the inherent variability of routinemicroscopic semen examination [27]. It simply involves thegrading of sperm motility into four orders, which are, highlywww.animbiosci.org1255
Tanga et al (2021) Anim Biosci 34:1253-1270progressively motile, progressively motile, non-progressivelymotile, and immotile (grade a, b, c, and d). While this presented breakthroughs for sperm-motility quantification, theywere largely black boxes with little true verification and skepticism.In domestic animals, commercial use of a fully automatedCASA system was introduced in 1985 [62]. In the beginning,the CASA systems utilized for sperm analysis were basedon an expert vision system developed for other purposes[63,64]. Later on, in 1992, old systems were replaced withautomated CASA systems specifically developed for determining sperm-morphology of bull [65], dog [66], human[67], and stallion [68]. Many CASA systems utilize differentfilters to better recognize sperm and represent great improvements. For streamline accuracy, intelligent filters form partof the software that can detect if a “particle” has a tail. Thisbecame an advantage in case of contaminated semen sampleswhich are difficult to analyze using other software.CASA generated motility data of sperm, (percentage andconcentration of motile sperm) which is very helpful for theprediction of fertility in humans in vivo [69] and in vitro [70].The development of CASA has enabled the evaluation ofvarious fertility parameters. The recent development of CASAtechnology to analyze key kinematic functionality parameterssuch as hyperactivation, enabling visualization of sperm inthree dimensions, and measuring the flagellar and spermtracking (FAST) in a quantitative way will be of paramountimportance to assess sperm characteristics and fertility indetail [62,71].However, the CASA system has some limitations, affectedby the higher sperm concentration, which interferes withprogressive motility [37,72,73]. During semen analysis, shortcomings such as low contrast images and artifacts of dirtcan negatively affect the accuracy of the CASA software. Insuch cases, sperm concentration is greatly overestimatedand the percentage motility of sperm is underestimated. Theseissues remained inherent in most CASA systems only to bepartly resolved in the last two decades by some CASA systems.Another problem associated with CASA motility was thatit was based on video frames associated with either NTSCor PAL systems (restrictive in frame rates), and poor resolution cameras that were very restrictive. Furthermore, forreliable measurements and generating unbiased data, a concentration between 20 to 50 106 sperm/mL has been recordedin some CASA studies [74-77].Kinematic parameters using computer-assisted spermanalysisIn addition to sperm motility and concentration, the CASAsystem can analyze different kinematic parameters includingcurvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR),1256www.animbiosci.orgwobble (WOB), beat cross frequency (BCF), and amplitudeof lateral head displacement (ALH). VCL analyzes the velocityof a sperm head on time-average basis along its actual curvilinear path (measured in in two dimensions). VSL measuresthe velocity of a sperm head on a time-average basis alongthe straight line between its first and last detected position.VAP measures the velocity of a sperm head along its averagepath on a time-average basis. This path is computed by smoothing the actual path according to algorithms in the CASAinstrument, these algorithms vary among instruments. ALHmeasures the magnitude of the lateral displacement of asperm head about its average path. This can be expressedas the maximum or an average of such displacement [69].LIN estimates a curvilinear path reflecting the straightnessof the sperm path (VSL/VCL 100 [%]); STR, reflecting therighteousness of motion (VSL/VAP 100 [%]); WOB, is thedegree of oscillation of the actual path of the sperm headin its relationship with VAP (VAP/VCL 100 [%]). The progression of sperm in CASA also measures LIN, righteousnessmovement or STR, and Balancing or WOB, expressed aspercentages.ALH and BCF are measurements derived from VAP, whereALH is the amplitude of variations of the current path of thesperm head in its relationship with VAP, and BCF is the average rate at which the actual sperm trajectory crosses the VAP(a derivation of the true frequency of flagellar beat and frequency of rotation of the head). Although, CASA is a newtechnology and most of the progression of the sperm measurements are derived from the speed/velocity measurement,we did not find studies showing their correlation with fertility.Despite this, we recommend further studies should be performed on how these measurements can be utilized forassessing sperm fertility, unless there is no need to measure.Rather than the PR, VAP is preferred to evaluate and predict the fertilizing potential of fresh or post-thawed bull semen[78]. The evaluation of sperm kinematic parameters withfertilizing capacity in bulls showed that all VCL, VSL, andVAP in post-thaw doses for AI were found to be correlatedin achieving pregnancy [78-80]. From amongst the three kinematic parameters, VCL, VSL, VAP, VAP shows the highestcorrelation with fertility, and it may be the most useful spermspeed/velocity parameter, which can be relied upon for theestimation of sperm fertility [78].Flagellar and sperm trackingThe FAST method analyzes the movement of sperm by usingthe FAST program. FAST measures the flagellar beat frequencyand the tangent and curvature of the flagellar wave to determine the arc-length (true length of the track). The analysis isusually conducted at higher frame rates, and tracks are represented quantitatively in three dimensions [71]. The FASTmeasures flagellar beat frequency, flagellar arc wave speed,
Tanga et al (2021) Anim Biosci 34:1253-1270flagellar arc wavelength (μm) (fAWL), Sperm flagellar length(μm), flagellar power dissipation tail length (fwatts), flagellarpower dissipation first 30 μm (fwatts30), and potentially, alsoamplitude of flagellar displacement measured in TCS trackcentroid speed (progressiveness) [62,71].Computer-assisted sperm analysis-three dimensions(3D) of sperm evaluationThe three dimensions (3D) system analyzes sperm tracks inthree dimensions (X, Y, and Z axes); Z-axis is reconstructed as the sperm of most animal species are swimming in aspherical helix [62,81-83]. However, the 3D method has thedisadvantage that the Z-axis is presumed to be harmonic,but in most cases, it is not possible. In the 3D patterns, thereare clear differences, such as, in a bull sperm with a muchhigher speed and ALH, the helix diameter is almost twicethat of boar sperm. A sperm of Saanen goat has similar VCLas that of a bull sperm, however, the BCF is an importantfactor here, showing small stepwise increases in the Z planein Saanen goat sperm, in comparison to the larger Z-planeincreases observed in bull sperm [62]. Although the 3D methodis useful in describing qualitative differences, it is not necessarily useful in describing quantitative differences. However,there is a need for further investigations to uncover the correlation between 3D measurements and fertility indicators.In combination, the findings of FAST and 3D can be usedfor the advanced analysis of sperm-motility, such as measurements of sperm capacitation and hyperactivity, whichwas not possible with the conventional approach. This canbe achieved by analyzing flagellar speed increase as well asenergy expenditure (in Watts) over the first 30 μm length ofthe flagellum. There is a decrease in sperm flagellar beat frequency as it becomes capacitated and hyperactivated, resultingin tumbling sperm which shows swimming star spin patterns[62]. In general, CASA-based sperm analysis is one of theeasiest, fastest, and reliable fertility assessments for semenevolution in individual animals, and it would be a method ofchoice for undertaking sperm evaluation comparative studies[84].The future of computer-assisted sperm analysisThe CASA analyzes sperm motility, which is directly correlated with fertility reported for bovine [85], equine [86], ovine[87], rabbit [88], and swine [89] sperm. What makes CASAessential is that it brought improvements in the quantitativeanalysis of the sperm quality in terms of accuracy and precision, as compared to conventional motility measurementmethods [90-92]. It should be noted that there are variousCASA system brands applicable for sperm evaluation, andstandardization of the methods is of paramount importanceto make objective measurements to precisely determine fertility [27,93].The results of CASA should be defined in terms of themeasurement conditions that are correlated with the outcomefigures. In particular, the rate of image acquisition, time fortracking sample, smoothing algorithm, sperm concentrationper sample, chamber’s type and depth, model and softwareversion, microscope optic and magnification should be clearlydefined to interpret CASA results to determine fertility indifferent species and conditions [94-97]. The setting of CASAmeasurements must be standardized for each species, elsethe instrument settings will affect the measurement results[27]. The results of CASA measurements can be interpretedinto different parameters of sperm characteristics, includingkinematic parameters, FAST, and 3D sperm evaluation.Over the decades, the CASA system has developed protocols to efficiently analyze the kinematic parameters ofsperm in animals [98,99]. However, its use is not just limitedto kinematic parameters, it can also analyze morphology,viability, DNA fragmentation, and acrosome reaction [100],which are considered vital for fertility determination. Computer-based semen analysis is striving to develop automaticmicroscopic systems by using artificial intelligence techniques(deep learning, machine learning, and computer vision).Sperm morphology needs to be better elaborated by including the entire cell and especially understanding the tailcharacteristics by polychromatic staining in CASA. Thereseems to be a need for agreement in the selection of stainsfor use in conjunction with CASA for tail-characteristicstudies. In future, developments could lead to no furtherneed for staining and looking at the possibilities of moredetailed analysis by using phase-contrast and Nomarskidifferential interference contrast optics. It is also expectedthat the CASA system will develop a new automatic analysisfor new tests such as reactive oxygen species (ROS), mitochondrial assay, sperm maturity, sperm chromatin packaging,3D reconstruction, and tail analysis. This will further enhanceour understanding of sperm functionality.PLASMA MEMBRANE INTEGRITYThe integrity of the plasma membrane is essential for propersperm function and fertility, pertaining to its function tomaintain homeostasis [101,102], protection against foreignagents, and interactions with other cells, including oocytes,and the epithelial lining of the female reproductive tract [103,104]. Sperm fertility is dependent on the integrity of theplasma membrane, as it plays an important role in differentphysiological events such as capacitation, acrosomal reaction,and zona binding [105].Plasma membrane integrity is usually analyzed for a viability test, as it is pivotal for communication with other cellsand the environment [106]. Methods used for analyzingsperm plasma membrane are normally based on the enhancedwww.animbiosci.org1257
Tanga et al (2021) Anim Biosci 34:1253-1270permeability of damaged membrane [107]. These methodsinclude the hypo-osmotic swelling (HOS) test [108,109],eosin–nigrosin staining [110,111], and use of fluorescentprobes [112,113]. Fluorescent probes such as Hoechst orpropidium-iodide (PI) are either used alone or in combinationwith other permeable fluorochromes such as carboxyfluorescein diacetate or SYBR [114-116]. Complementary techniques,including flow cytometry and fluorimetry, can be helpfulin improving measurement precision, as large numbers offluorochrome-stained sperm can be assessed in a short duration of time [117,118]. The recent development of theCASA technique has allowed the automated measurementof sperm plasma membrane integrity [119].VIABILITY AND ACROSOMAL STATUSSperm viability is a key factor for quality analysis and a prerequisite for success in fertilization, particularly in cases, suchas AI, where low sperm numbers are used [120]. Eosin–nigrosin has been conventionally employed as a differentialstain to assess the proportion of live and dead sperm. Eosin,being the cellular stain, stains the dead sperm with the damaged plasma membrane, whereas the live sperm will not attainany color and remain colorless and the nigrosin stains thebackground. In addition, several other stains have been widelyused for sperm viability assessment, including fast–greenand eosin, and opal–blue and eosin [121]. However, none ofthese methods has yielded consistently satisfactory results incomparison to that of eosin–nigrosin. Recently, sperm viability has been assessed using a combination of molecularprobes, including SYBR-14 and PI [112]. After SYBR-14 andPI staining, live sperm percentage was determined by fluorescence microscopy, while flow cytometry was used to assessthe uptake of stain. Live sperm are stained green with SYBR14, whereas the dead sperm are stained red by PI.Acrosomal integrity is one of the determining factors forfertility because, for successful fertilization, sperm must havean intact acrosome and must react on time when they reachthe site of fertilization [122-124]. The ideal test used for theanalysis of sperm acrosomal status should be precise, quick,applicable to samples with low sperm count, safe for spermfunction, and must be able to differentiate normal from falseacrosome reactions [125]. Previously, the acrosomal integrityof spermatozoa was assessed by fixing the sperm sample in1% formal citrate solution [126]. Sperm with normal acrosomes
Semen primarily consists of two main components: the sperm and seminal fluids; sperm * Corresponding Author: Jongki Cho . plasma membrane and genomic constituents, and enables better deter
Sexed semen technology in cattle: A revolutionary technique in Indian dairy industry Dharmveer Singh, Pramod Kumar, KS Nehra and Ajay Kumar Abstract Desired sex either female or male which is produce from semen having X or Y bearing sperm is known as sexed semen. Sexed semen increase the
PT SEMEN INDONESIA (PERSERO) Tbk DAN ENTITAS ANAK LAPORAN POSISI KEUANGAN KONSOLIDASIAN 31 Maret 2019 dan 31 Desember 2018 (Disajikan dalam ribuan Rupiah, kecuali dinyatakan lain) PT SEMEN INDONESIA (PERSERO) Tbk AND ITS SUBSIDIARIES CONSOLIDATED STATEMENTS OF FINANCIAL POSITION March 31, 2019 and December 31, 2018 (Expressed in thousands of .
1.2 The fi fth edition 1 1.3 Scope of the manual 3 PART I. SEMEN ANALYSIS Chapter 2 Standard procedures 7 2.1 Introduction 7 2.2 Sample collection 10 2.2.1 Preparation 10 2.2.2 Collection of semen for diagnostic or research purposes 11 2.2.3 Sterile collection of semen for assisted reproduction 11
8 Laporan Keberlanjutan 2013 PT SEMEN PADANG Laporan Keberlanjutan 2013 PT SEMEN PADANG 9 LAYANAN PELANGGAN Merealisasikan 52 item kegiatan promosi above and below the line sebagai salah satu persiapan ekspansi produk di wilayah pemasaran. Merealisasikan program survey kepuasan pelanggan (Customer Satisfaction Index/
Financial Statements Annual Report Award 2016 Criteria 280 Annual Report Laporan Tahunan 2016 PT SEMEN TONASA Annual Report Laporan Tahunan 2016 PT SEMEN TONASA 281 Perseroan memandang bahwa Sumber Daya Manusia (SDM) merupakan komponen yang sangat penting dalam rangka mempertahankan keung
MALE INFERTILITY AND SEMEN PARAMETERS: A DEMYSTIFIED REVIEW ERA’S JOURNAL OF MEDICAL RESEARCH, VOL.7 NO.1 Page: 112 Parameter Lower reference limit Semen volume (ml) 1.5 (1.4-1.7) Total sperm number (106 per ejaculat
the USA.Launch of Sacol, the on-farm AI Straw, for swine semen packaging. Launch of the TBS Straw for Bovine Semen Production: a productivity gain for semen production laboratories. 2002 Opening of IMV China, IMV Technologies’ representative offi
Article 505. Class I, Zone 0, 1, and 2 Locations Figure 500–2. Mike Holt Enterprises, Inc. www.MikeHolt.com 888.NEC.CODE (632.2633) 25 Hazardous (Classified) Locations 500.4 500.4 General (A) Classification Documentation. All hazardous (classified) locations must be properly documented. The documentation must be available to those who are authorized to design, install, inspect .
