Ultrasonographic measures of body fatness and their relationship with plasma levels and adipose tissue expression of four adipokines in Welsh pony mares.

Obesity is responsible for metabolic dysregulations that alter fertility and induce pathologies. The objectives of the present study were to validate a reliable method for the evaluation of body fatness in mares and to associate the body fat estimation data to metabolic changes, including adipokines at the plasma and adipose tissue levels. To reach this purpose, animals were subjected to two extreme breeding conditions to study the variation of morphological, ultrasound, and physiological parameters. Twenty Welsh mares were followed up monthly from April to October before and after animals were moved outdoors to grasslands. Body weight (BW), body length (BL), height at the withers (HW), thoracic perimeter (TP), 5-point body condition score (BCS), and subcutaneous fat thickness (SFT) at the level of the shoulder, the lumbar region, and the rump, measured by ultrasonography, and plasma and adipose tissue metabolic indicators were assessed in parallel. Statistical analysis was performed using a linear mixed-effects model, whereas Pearson tests were used for the analysis of the correlations between the different parameters. Although mean BW did not increase significantly (P = 0.0940), TP (P = 0.0002) and BCS (P < 0.0001) increased during the study period. Ultrasonographic examination of subcutaneous adipose tissue showed an increase in SFT at the level of the shoulder (P < 0.0001), lumbar region (P < 0.0001), and rump (P < 0.0001). Plasma concentrations of nonesterified fatty acids (P < 0.0001), phospholipids (P < 0.0001), and cholesterol (P < 0.0001) increased significantly, whereas triglycerides (P < 0.0001) decreased significantly during the study period. Although both plasma concentrations and adipose tissue expression of leptin (P < 0.0001) and resistin (P < 0.0001) increased significantly, adiponectin (P < 0.0001) significantly decreased and visfatin remained unchanged (P = 0.8401). Expression of adipokine receptors studied showed the opposite pattern compared with their ligand. Ultrasonographic measurements of subcutaneous adipose tissue thickness at the shoulder, lumbar region, and rump are relevant indicators of fatness related with adipokine plasma concentrations and expression of adipokine-related receptors in adipose tissue, and particularly highlight seasonal effects.

Salivary and urinary metabolome analysis for pre-puberty-related biomarkers identification in porcine.

Estrus synchronization is important for optimal management of gilt reproduction in pig farms. Hormonal treatments, such as synthetic progestogens, are used on a routine basis, but there is a growing demand for non-hormonal alternative breeding tools. Before puberty, gilts exhibit a 'waiting period,' related to the ovarian development and gonadotrophin secretions, during which external stimulations, such as boar exposure, could induce and synchronize first ovulation. Practical non-invasive tools for identification of this period in farms are lacking. During this period, urinary oestrone levels are high, but urine sampling is difficult in group-housed females. The aim of this work was to search for specific biomarkers of the 'waiting period' in saliva and urine. In total, nine 144- to 147-day-old Large White gilts were subjected to trans-abdominal ultrasonography three times a week for 5 weeks until puberty detection (week -5 to week -1 before puberty). Urine and saliva samples were collected for oestrone assay to detect the 'waiting period' and for metabolome analysis using 1H-nuclear magnetic resonance spectroscopy to detect potential biomarkers of the 'waiting period.' Gilts were slaughtered 7 days after puberty detection for puberty confirmation. Results were consistent with ultrasonography data for six gilts. Urine and saliva samples from these six gilts were analyzed. Urinary estrone concentration significantly increased 2 weeks before puberty detection. Metabolome analysis of urine samples allowed the identification of 78 spectral bins, among them, 42 low-molecular-weight metabolites were identified. Metabolome analysis of salivary samples allowed the identification of 59 spectral bins, among them, 23 low-molecular-weight metabolites were detected and 17 were identified. No potential biomarker was identified in urinary samples. In saliva, butyrate and 2HOvalerate, 5.79 ppm (putatively uridine), formate, malonate and propionate could be biomarker candidates to ascertain the pre-puberty period in gilt reproduction. These results confirm that non-invasive salivary samples could allow the identification of the physiological status of the gilts and presumably the optimal time for application of the boar effect. This could contribute to synchronize puberty onset and hence to develop non-hormonal breeding tools.

Role of early life nutrition on regulating the hypothalamic­anterior pituitary­testicular axis of the bull

The objective of this study was to examine the effect of nutrition during the first 18 weeks of life on the physiological and transcriptional functionality of the hypothalamic (arcuate nucleus region), anterior pituitary and testes in Holstein­Friesian bull calves. Holstein­Friesian bull calves with a mean (±S.D.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were assigned to either a HIGH (n = 10) or LOW (n = 10) plane of nutrition, to achieve an overall target growth rate of 1.2 or 0.5 kg/day, respectively. At 126 ± 1.1 days of age, all calves were euthanised. Animal performance (weekly) and systemic concentrations of metabolic (monthly) and reproductive hormones (fortnightly) were assessed. Testicular histology, targeted gene and protein expression of the arcuate nucleus region, anterior pituitary and testes were also assessed using qPCR and immunohistochemistry, respectively. The expression of candidate genes in testicular tissue from post pubertal 19-month-old Holstein­Friesian bulls (n = 10) was compared to that of the 18-week-old calves. Metabolite and metabolic hormone profiles generally reflected the improved metabolic status of the calves on the HIGH (P< 0.001). Calves offered a HIGH plane of nutrition were heavier at slaughter (P < 0.001), had larger testes (P < 0.001), larger seminiferous tubule diameter (P < 0.001), more mature spermatogenic cells (P < 0.001) and more Sertoli cells (P < 0.05) in accordance with both morphological and transcriptional data. Overall, testicular gene expression profiles suggested a more mature stage of development in HIGH compared with LOW and were more closely aligned to that of mature bulls. Ghrelin receptor was the only differentially expressed gene between LOW and HIGH calves in either the anterior pituitary (P < 0.05) or arcuate nucleus region of the hypothalamus (P < 0.10) and was upregulated in LOW for both tissues. This study indicates that an enhanced plane of nutrition during early calfhood favourably alters the biochemical regulation of the hypothalamus­anterior pituitary­testicular axis, advancing testicular development and hastening spermatogenesis.

Review: Spermatogenesis in the bull.

Spermatogenesis is a finely regulated process of germ cell multiplication and differentiation leading to the production of spermatozoa in the seminiferous tubules. Spermatogenesis can be divided into three parts: spermatocytogenesis, meiosis and spermiogenesis. During spermatocytogenesis, germ cells engage in a cycle of several mitotic divisions that increases the yield of spermatogenesis and to renew stem cells and produce spermatogonia and primary spermatocytes. Meiosis involves duplication and exchange of genetic material and two cell divisions that reduce the chromosome number and yield four haploid round spermatids. Spermiogenesis involves the differentiation of round spermatids into fully mature spermatozoa released into the lumin of seminiferous tubules. The seminiferous epithelium is composed of several generations of germ cells due to the fact that new generations of sperm cells engage in the spermatogenic process without waiting for the preceding generations to have completed their evolution and to have disappeared as spermatozoa into the lumen of the tubules. In bulls, the duration of the seminiferous epithelium cycle is 13.5 days. The total duration of spermatogenesis is 61 days, that is 4.5 times the duration of the cycle of the seminiferous epithelium. The spermatogenetic wave is used to describe the spatial arrangement of cell associations along the tubules. Several theories have been described to explain the renewal of spermatogonia. Depending on the model, there are five or six spermatogonial mitoses explaining the renewal of stem cells and the proliferation of spermatogonia. Daily sperm production and germ cell degeneration can be quantified from numbers of germ cells in various steps of development throughout spermatogenesis. Bulls have a lower efficiency of spermatogenesis than most species examined, but higher than that of humans.

Plane of nutrition before and after 6 months of age in Holstein-Friesian bulls: I. Effects on performance, body composition, age at puberty, and postpubertal semen production.

The aim of this study was to examine the effect of plane of nutrition (1) during the first 6 mo of life and (2) from 6 mo of age to puberty on early growth characteristics, age at puberty, and postpubertal semen production in Holstein-Friesian bulls. Holstein-Friesian bull calves (n = 83) with a mean (standard deviation) age and body weight of 17 (4.4) d and 52 (6.2) kg, respectively, were assigned to a high (Hi) or low (Lo) plane of nutrition for the first 6 mo of life. The Hi and Lo calves received 1,200 and 450 g of milk replacer, respectively; Hi calves were fed concentrate ad libitum and Lo were fed a maximum of 1 kg concentrate daily, and concentrate allowances remained the same after weaning. At 24 wk of age, bulls were reassigned within treatment to either remain on the same diet or to switch to the opposite diet until puberty, resulting in 4 treatment groups: Hi-Hi, Hi-Lo, Lo-Lo, and Lo-Hi. After puberty, all bulls were fed a moderate plane of nutrition until 60 wk of age; thereafter, the diet was ad libitum concentrates until slaughter at 72 wk of age. Bulls were weighed weekly before weaning and every 2 wk after weaning. Scrotal circumference (SC) was measured every 2 wk, beginning at 15 wk of age. Beginning at a SC of 24 cm, electro-ejaculation was carried out every 2 wk to establish the onset of puberty. Semen collection continued monthly after puberty. Thermal images of the scrotum were taken monthly from 28 to 36 wk of age. Scrotal skin thickness (SST) was measured monthly (from 16 wk of age to puberty) using a digital calipers. Bulls on the Hi diet had a higher scrotal temperature and SST at each time point than those on the Lo diet. Average daily gain (ADG) was greatest in Hi-Hi bulls, with Hi-Lo and Lo-Hi having similar ADG but both being greater than Lo-Lo. Bulls on the Hi diet pre-6 mo of age were younger at puberty, regardless of diet offered post-6 mo of age. Bulls offered a Hi diet post-6 mo were heavier at puberty. Neither scrotal temperature nor dietary treatment affected postpubertal semen production variables. In conclusion, a high plane of nutrition during the first 6 mo of age hastened the onset of puberty and the availability of saleable semen, regardless of plane of nutrition post-6 mo of age.

Large Independent Primary Care Medical Groups.

PURPOSE: In the turbulent US health care environment, many primary care physicians seek hospital employment. Large physician-owned primary care groups are an alternative, but few physicians or policy makers realize that such groups exist. We wanted to describe these groups, their advantages, and their challenges. METHODS: We identified 21 groups and studied 5 that varied in size and location. We conducted interviews with group leaders, surveyed randomly selected group physicians, and interviewed external observers-leaders of a health plan, hospital, and specialty medical group that shared patients with the group. We triangulated responses from group leaders, group physicians, and external observers to identify key themes. RESULTS: The groups' physicians work in small practices, with the group providing economies of scale necessary to develop laboratory and imaging services, health information technology, and quality improvement infrastructure. The groups differ in their size and the extent to which they engage in value-based contracting, though all are moving to increase the amount of financial risk they take for their quality and cost performance. Unlike hospital-employed and multispecialty groups, independent primary care groups can aim to reduce health care costs without conflicting incentives to fill hospital beds and keep specialist incomes high. Each group was positively regarded by external observers. The groups are under pressure, however, to sell to organizations that can provide capital for additional infrastructure to engage in value-based contracting, as well as provide substantial income to physicians from the sale. CONCLUSIONS: Large, independent primary care groups have the potential to make primary care attractive to physicians and to improve patient care by combining human scale advantages of physician autonomy and the small practice setting with resources that are important to succeed in value-based contracting.

Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots.

The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session.

Oral flurbiprofen metabolic ratio assessment using a single-point dried blood spot.

We investigated whether a single blood measurement using the minimally invasive technique of a finger prick to draw a blood sample of 5 µl (to yield a dried blood spot (DBS)) is suitable for the assessment of flurbiprofen (FLB) metabolic ratio (MR). Ten healthy volunteers who had been genotyped for CYP2C9 were recruited as subjects. They received FLB alone in session 1 and FLB with fluconazole in session 2. In session 3, the subjects were pretreated for 4 days with rifampicin and received FLB with the last dose of rifampicin on day 5. Plasma and DBS samples were obtained between 0 and 8 h after FLB administration, and urine was collected during the 8 h after administration. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. FLB's apparent clearance values decreased by 35% in plasma and DBS during session 2 and increased by 75% in plasma and by 30% in DBS during session 3. Good correlations were observed between MRs calculated from urine, plasma, and DBS samples.

Is photometry an accurate and reliable method to assess boar semen concentration?

Sperm concentration assessment is a key point to insure appropriate sperm number per dose in species subjected to artificial insemination (AI). The aim of the present study was to evaluate the accuracy and reliability of two commercially available photometers, AccuCell™ and AccuRead™ pre-calibrated for boar semen in comparison to UltiMate™ boar version 12.3D, NucleoCounter SP100 and Thoma hemacytometer. For each type of instrument, concentration was measured on 34 boar semen samples in quadruplicate and agreement between measurements and instruments were evaluated. Accuracy for both photometers was illustrated by mean of percentage differences to the general mean. It was -0.6% and 0.5% for Accucell™ and Accuread™ respectively, no significant differences were found between instrument and mean of measurement among all equipment. Repeatability for both photometers was 1.8% and 3.2% for AccuCell™ and AccuRead™ respectively. Low differences were observed between instruments (confidence interval 3%) except when hemacytometer was used as a reference. Even though hemacytometer is considered worldwide as the gold standard, it is the more variable instrument (confidence interval 7.1%). The conclusion is that routine photometry measures of raw semen concentration are reliable, accurate and precise using AccuRead™ or AccuCell™. There are multiple steps in semen processing that can induce sperm loss and therefore increase differences between theoretical and real sperm numbers in doses. Potential biases that depend on the workflow but not on the initial photometric measure of semen concentration are discussed.

Proteome analysis and genome-wide regulatory motif prediction identify novel potentially sex-hormone regulated proteins in rat efferent ducts.

The efferent ducts are a series of tubules that conduct sperm from the rete testis to the epididymis. They absorb most fluid and proteins originating from the rete testis during concentration of spermatozoa prior to their entry into the epididymis. Proteome analysis of micro-dissected efferent duct samples from adult rats was combined with genome-wide computational prediction of conserved hormone response elements to identify factors likely regulated by oestrogens and androgens. We identified 165 proteins and found subsets of the promoters controlling their corresponding genes to contain androgen- and oestrogen response elements (ARE/EREs) at similar frequencies. Moreover, EREs were significantly enriched among the loci identified compared with their genome-wide occurrence. The expression and localization of Anxa6, Ckb, Krt19, Park7, Pdzk1 and Tpt1 in the efferent ducts and other related hormone controlled tissues was further validated at the RNA or protein level. This study identifies many novel proteins predicted to play roles in sperm maturation and male fertility and provides significant computational evidence that the efferent ducts express genes transcriptionally controlled by sex hormones.

[Ethyl glucuronide: a biomarker of alcohol consumption]. / L'éthylglucuronide: un marquer de la consommation d'alcool.

Excessive alcohol consumption represents a major risk factor for morbidity and mortality. It is therefore indispensable to be able to detect at-risk drinking. Ethyl glucuronide (EtG) is a specific marker of alcohol consumption. The determination of ethyl glucuronide in urine or blood can be used to prove recent driving under the influence of alcohol, even if ethanol is no longer detectable. The commercialization of an EtG specific immunological assay now allows to obtain preliminary results rapidly and easily with satisfying sensitivity. Moreover, the detection of ethyl glucuronide in hair offers the opportunity to evaluate an alcohol consumption over a long period. The EtG concentration in hair is in correlation with the amount of ingested alcohol. Thus, the analysis of ethyl glucuronide can be used to monitor abstinence, to detect alcohol relapse and to identify at-risk drinkers. However, a cut off allowing to detect chronic alcohol abuser reliably still does not exist. Therefore, it is recommended to perform the analysis of ethyl glucuronide in complement to the existing blood markers. A study financed by the Swiss Foundation for Alcohol Research is actually conducted by the West Switzerland University Center of Legal Medicine in order to establish an objective cut-off.

Hair analysis to document a clinical case of TCDD over-exposure.

Dioxins and related compounds (furans) are persistent environmental contaminants that cause adverse biological effects. Their influence on humans is still unclear, except for accidental high-dose exposure. Chronic exposure to these compounds seems to be involved in cancer, endocrine disruption and neurobehavioral effects. For several years, a large concern about the potential health risks of dioxins is emerging in Europe and United States. The case of a 50-year-old man victim of an acute over-exposure to tetrachloro dibenzo-p-dioxin (TCDD or Seveso dioxin) is reported here with particular focus on hair investigations. The developed method involved the decontamination of the hair strand using picograde level methylene chloride, the homogenization of hair segments with scissors and their extraction in presence of 13C12-marked dioxin congeners under reflux of toluene using a Soxhlet, 8h at 130 degrees C. After reduction of the toluene fraction to 1 ml and addition of purification marker (37Cl4-2,3,7,8-TCDD), dioxins purification was achieved using three successive columns: silica, alumina/sodium sulfate and carbon/Celite columns. Finally, the toluene eluent was evaporated and the extract injected in the analytical system. After chromatographic separation, detection was achieved in single ion monitoring mode using a high resolution mass spectrometer operating in electron impact ionization mode (40 eV, minimal resolution of 10,000). The analysis of the first hair segment (0-6 cm) revealed the presence of 2,3,7,8-TCDD at 65 fg/mg when the distal one remained negative (LOQ=0.3 fg/mg). All other congeners (n=16) were in the range of those determined in the general population (0.62 and 0.89 fg/mg in the two hair segments, respectively). The extremely low dioxin levels generally found in hair specimens (low fg/mg range) lead us to analyze them using the very sensitive and specific gas chromatography-high resolution mass spectrometry apparatus. From the best of our knowledge, this is the first case of over-exposure to Seveso dioxin through hair analysis reported in the literature.

Transferrin overexpression alters testicular function in aged mice.

Many studies have shown a correlation between transferrin (Tf) concentration and sperm yield in several mammalian species. We have used transgenic mice expressing human Tf (hTf) to investigate if overexpression of Tf increases the efficiency of mouse spermatogenesis. We demonstrated that a 36% increase of Tf does not ameliorate the efficiency of mouse spermatogenesis but on the contrary resulted in a 36% decrease of testis sperm reserves. Tf overexpression had no effect on testicular determination and development, however testicular function of these transgenic mice was affected in an age-dependent manner. At 16 months of age, testicular and epididymal weights were significantly reduced. While spermatogenesis was qualitatively normal, testicular functions were perturbed. In fact, testosterone rate after human chorionic gonadotropin (hCG) stimulation was lower in Tf overexpressing mice. Intratesticular concentration of estradiol-17beta was increased and fluid accumulation after ligation of rete testis was more abundant in these transgenic mice. Surprisingly, we found that endogenous Tf levels were also increased in Tf overexpressing mice and we demonstrated for the first time that Tf may serve to upregulate its own expression in testis. Collectively, our data show that Tf overexpression has negative effects on testicular function and that Tf levels require strict regulation in the testis.

[The comparison of the process of five different vitrification devices]. / Principe de la vitrification: cinétiques comparatives.

Over the last decade, several methods have been designed to improve the survival rate of vitrificated embryos. Although some teams have succeeded, the main remaining drawback of these methods is that they do not provide a leak proof environment for cryopreserved biological samples. To respond to that demand in respect with the European reglementation, the Cryo Bio System company (CBS) designed the HSV High Security Vitrification Kit (HSV). This system is composed of three distinct parts, a High Security thermal-autogenic sealed clear straw, a capillary with its extremity in form of a gutter, and an introducer that can be mounted on the manipulation rod before introduction into the straw. In this study, we confirmed that the CBS vitrification kit is a suitable method for vitrification in association with a small amount of cryoprotector enriched viscous media such as 25 microM Ficool 400, 750 mM Sucrose, 1% Bovine albumin, 20% Dimethyl Sulfoxide and 20% Ethylene glycol in a Phosphate buffered saline solution. We also evaluated the speed of the temperature decrease during vitrification in comparison with four other commercially available non-aseptic methods and showed the protective role of the CBS system during transfer. These physical data have now been confirmed biologically by P. Vanderzwalmen who obtained easily reproductible good results with human embryo using our method. Today, the HSV represents the unique aseptic alternative device (EC and FDA approved) for embryos, oocytes, and biological samples vitrification.

Human testis in organotypic culture: application for basic or clinical research.

BACKGROUND: Over recent decades, recurring efforts have been devoted to developing testicular cell or tissue cultures for basic and clinical research. However, there remains much confusion, particularly concerning the fate of human germ cells in culture. OBJECTIVE: To reassess the status of human testicular cell types as well as the ability of germ cells to divide and differentiate in organotypic culture. METHODS: Human testicular fragments were maintained for 2 weeks in culture. The viability and functionality of testicular cells were assessed using light and electronic microscopy, apoptotic cell labelling, 5-bromo-2'-deoxyuridine (BrdU) incorporation, immunohistochemistry and quantitative PCR against specific cell markers. RESULTS: A gradual loss of meiotic and post-meiotic germ cells occurred throughout the culture period, irrespective of the presence of gonadotrophins. However, all germ cell types remained traceable for up to 16 days, some still dividing and differentiating at a rate compatible with the in vivo situation. Good maintenance of the general architecture of the explants associated with clearly quantifiable levels of several somatic cell markers was observed. CONCLUSION: Although this culture model is clearly unsuitable for preparing germ cells for therapeutic purposes, it does represent a most valuable tool for testing the effects of biological and chemical agents on testicular tissue.

Determination of benzodiazepines in human hair by on-line high-performance liquid chromatography using a restricted access extraction column.

A method is described for the identification of five frequently prescribed benzodiazepines (BZD) (clonazepam, diazepam, flunitrazepam, midazolam and oxazepam) in human hair samples by reversed phase HPLC, following on-line simple enrichment and clean-up on a restricted access extraction column. 50mg of powdered hair were incubated (2h at 45 degrees C) after sonication (1h) in 1 ml of the following solution (methanol:ammonia, 97.5/2.5, v/v). The aliquot was centrifuged and the methanolic phase transferred to a conical tube and evaporated under a gentle stream of nitrogen. The residue was reconstituted by adding 100 microl of a mixture of phosphate buffer (20mM, pH=2.2) and acetonitrile (94/6, v/v). A total of 80 microl were injected into the system with the column switching technique. The pre-column or clean-up column was washed with phosphate buffer pH=7.2. The drugs retained on the pre-column were then eluted in the back-flush mode and separated on a C(8) semi micro column, Lichrospher select B, 125 mm x 3 mm. The BZD were determined by a photodiode-array detector at 254 nm, using reference data (retention time and UV spectra) stored in a personal library. The method showed excellent linearity between 0.5 and 20 ng/mg of hair for clonazepam, flunitrazepam and midazolam and between 0.5 and 100 ng/mg of hair for diazepam and oxazepam. Finally, the present method has been applied to a number of forensic cases in our laboratory.

Live human germ cells in the context of their spermatogenic stages.

BACKGROUND: Various types of live, dispersed, human testicular cells in vitro were previously compared with the morphologic characteristics of human spermatogenic germ cells in situ within seminiferous tubules. The current study extends those observations by placing live human germ cells in the context of their developmental steps and stages of the spermatogenic cycle. METHODS: Live human testicular tissue was obtained from an organ-donating, brain-dead person. A cell suspension was obtained by enzymatic digestion, and dispersed cells were observed live with Nomarski optics. Testes from 10 men were obtained at autopsy within ten hours of death, fixed in glutaraldehyde, further fixed in osmium, embedded in Epon, sectioned at 20 microm, and observed unstained by Nomarski optics. RESULTS: In both live and fixed preparations, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells. For germ cells, size, shape, and chromatic pattern of nuclei, the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece are characteristically seen in live dispersed cells and those in the fixed seminiferous tubules. These lead to identification of live germ cells in man and placement of each in the context of their developmental steps of spermatogenesis at corresponding stages of the spermatogenic cycle. CONCLUSIONS: This comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells to be used in clinical procedures or by scientists interested in studying live cells at known steps in spermatogenic development characteristic of germ cells in specific stages of the spermatogenic cycle.