CpG DNA methylation changes during epididymal sperm maturation in bulls.

BACKGROUND: During epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions. RESULTS: Caput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda. CONCLUSIONS: Specific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

Intramammary administration of platelet concentrate as an unconventional therapy in bovine mastitis: first clinical application.

Bovine udder infections induce a variety of changes in gene expression of different growth factors that may suggest their possible role in glandular tissue protection or repair processes. Growth factors and also chemokines and cytokines may act synergistically to increase the infiltration of neutrophils and macrophages to promote angiogenesis, fibroplasia, matrix deposition, and, ultimately, re-epithelialization. Considering the vast applications, typically in human medicine, of platelet concentrate (PC) and its ease of preparation, the aim of our study was to evaluate an alternative therapy to stimulate the regeneration of glandular tissue, administering a concentration in excess of the growth factors contained in the PC. In each one of the 3 farms examined in the trial, PC was prepared from donor cows in good health, free from infections, and with no records of medications administered during the previous 2 mo. The platelet produced in one farm was used only for treating the cows of the same farm in a heterologous way. A total of 229 mastitic quarters were divided in 3 groups: antibiotic group (treated with intramammary antibiotic), antibiotic and PC group (treated intramammarily with antibiotics in association with PC), and PC group (treated with intramammary PC alone). The diagnosis of mastitis was based on somatic cell count and bacteriological evaluation of the milk from the affected quarter. Platelet concentrate, alone or in association with antibiotic, was used for 3 consecutive days as an unconventional therapy in bovine acute and chronic mastitis. Our data show that the associated action of antibiotic and PC performed significantly better than the antibiotic alone, either for the recovery of the affected mammary quarters or for somatic cell count reduction. In the same way, the association antibiotic plus PC showed significantly fewer relapses compared with the antibiotic alone, either for acute or chronic mastitis. The treatment with only PC did not show statistically significant differences compared with both antibiotic alone or associated treatment for acute mastitis, and it was better than the use of only antibiotic for chronic mastitis. Our results show that PC alone may be useful for a quick resolution of the inflammatory response, playing a role in limiting the tissue damage to the mammary gland parenchyma and reducing the recurrence rates.

Characteristics of equine mesenchymal stem cells derived from amnion and bone marrow: in vitro proliferative and multilineage potential assessment.

REASONS FOR PERFORMING STUDY: This is the first study comparing stemness features of equine mesenchymal progenitor cells derived from amniotic membrane and bone marrow. OBJECTIVES: To investigate an alternative and noninvasive stromal cell source for equine tissue engineering. STUDY DESIGN: In vitro experimental study of the characteristics of equine mesenchymal progenitor cells derived from amnion and bone marrow. METHODS: Cells isolated from amniotic membrane and bone marrow were analysed for proliferation (growth curve, doubling time, colony forming unit). Immunocytochemical detection of pluripotency markers and gene expression of stromal cell markers were also performed and these cells were studied for multilineage plasticity. RESULTS: Amniotic stromal cells (AMSCs) and bone marrow mesenchymal cells (BM-MSCs) both exhibited mature stromal cell-specific gene expression and immunocytochemical properties, but showed substantial differences in their proliferative and differentiation potential. The mean doubling time for AMSCs was significantly lower (P<0.05) than that observed for BM-MSCs (1.17 ± 0.15 vs. 3.27 ± 0.19 days, respectively). Compared to AMSCs, BM-MSCs also demonstrated a significantly (P<0.05) lower clonogenic capability (one fibroblast-like colony forming unit from a mean of 590.15 cells seeded for BM-MSCs vs. 242.73 cells seeded for AMSCs). BM-MSCs did not differentiate into glial cells, and the osteogenic differentiation process was longer than for AMSCs. CONCLUSIONS AND POTENTIAL RELEVANCE: The amniotic membrane could be a valuable source of MSCs to be used both for allogenic and/or autologous therapies. The noninvasive nature and low cost of collection, the rapid proliferation along with a greater differentiation potential and the 'off the shelf' preparation potential could make AMCs useful for cell therapy.

Mesenchymal stem cells from amnion and amniotic fluid in the bovine.

Amnion and amniotic fluid (AF) are noncontroversial and inexhaustible sources of mesenchymal stem cells (MSCs) that can be harvested noninvasively at low cost. As in humans, also in veterinary field, presumptive stem cells derived from these tissues reveal as promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. The aim of this work is to obtain and characterize, for the first time in bovine species, presumptive MSCs from the epithelial portion of the amnion (AECs) and from the AF (AF-MSCs) to be used for clinical applications. AECs display a polygonal morphology, whereas AF-MSCs exhibit a fibroblastic-like morphology only starting from the second passage, being heterogeneous during the primary culture. For both lines, the proliferative ability has been found constant over the ten passages studied and AECs show a statistically lower (P<0.05) doubling time with respect to AF-MSCs. AECs express MSC-specific markers (ITGB1 (CD29), CD44, ALCAM (CD166), ENG (CD105), and NT5E (CD73)) from P1 to P3; in AF-MSCs, only ITGB1, CD44, and ALCAM mRNAs are detected; NT5E is expressed from P2 and ENG has not been found at any passage. AF-MSCs and AECs are positive for the pluripotent markers (POU5F1 (OCT4) and MYC (c-Myc)) and lack of the hematopoietic markers. When appropriately induced, both cell lines are capable of differentiating into ectodermal and mesodermal lineages. This study contributes to reinforce the emerging importance of these cells as ideal tools in veterinary medicine. A deeper evaluation of the immunological properties needs to be performed in order to better understand their role in cellular therapy.

Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term extenders.

The aim of this study was to assess the effect on boar spermatozoa quality of in vitro storage in short and long-term extenders by fluorescent multiple staining (FMS) and computer assisted semen analyzer (CASA). Fresh ejaculates from three healthy, sexually mature boars were diluted with equal volumes of six short-term or three long-term commercial extenders and stored at 19°C for 6 days (short-term) or 12 days (long-term). The integrity of spermatozoa membranes was analyzed by FMS using propidium iodide, 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) and fluorescein isothiocyanate-conjugated peanut agglutinin (PNA). The results obtained from this staining were compared with spermatozoa motility assessed by CASA. Our study showed that the number of viable spermatozoa with non-reacted acrosomes and intact mitochondria was positively correlated with the rate of motile spermatozoa (r(2)>0.9) irrespective of the extender used. In all extenders the number of motile spermatozoa significantly decreased as preservation period increased (P<0.05). FMS test is a potent indicator of sperm motility because it analyses mitochondrial integrity independently from observable alterations in motility. The best performing extenders were BTS for short-term storage and TRI-x-Cell for long-term storage.

Characterization and potential applications of progenitor-like cells isolated from horse amniotic membrane.

The aim of this work was to isolate, for the first time, progenitor-like cells from the epithelial (AECs) and mesenchymal (AMCs) portions of the horse amniotic membrane, and to define the biological properties of these cells. AECs displayed polygonal epithelial morphology, while AMCs were fibroblast-like. Usually, six to eight passages were reached before proliferation decreased, with 13.08 and 26.5 cell population doublings attained after 31 days for AECs and AMCs, respectively. Immunocytochemical studies performed at passage 3 (P3) showed that both cell populations were positive for the expression of specific embryonic markers (TRA-1-60, SSEA-3, SSEA-4 and Oct-4). Meanwhile, RT-PCR performed at P1 and P5 showed expression of mesenchymal stem/stromal cell markers (CD29, CD105, CD44 and CD166) with negativity for CD34 at P1, although this marker began to be expressed by P5. The cells also expressed MHC-I at both P1 and P5, but lacked MHC-II expression at P1. Both AECs and AMCs demonstrated high plasticity, differentiating in vitro toward the osteogenic, adipogenic, chondrogenic and neurogenic lineages. Equine amnion-\derived cells could also be frozen and recovered without loss of their functional integrity in terms of morphology, presence of specific stemness markers and differentiation ability, although the renewal capacity was lower than that observed for freshly isolated cells. To investigate potential therapeutic effects and cell tolerance in vivo, horse amnion-derived cells were allogeneically injected into three horses with tendon injuries, resulting in a quick reduction in tendon size and ultrasonographic cross-sectional area measurements. These results suggest that horse amnion-derived cells may be useful for cell therapy applications.

Characterization and differentiation of equine tendon-derived progenitor cells.

Mesenchymal stem cells have been recently investigated for their potential use in regenerative medicine. Population of adult stem cells were recently identified in human and lab animal tendons, but no detailed investigations have been made in the equine species. The aim of our study is to identify a progenitor cell population from tendon tissue (TSPCs) in the horse superficial digital flexor tendon that are able to be highly clonogenic, to grow fast and to differentiate in different induced cell lineages as well as bone marrow derived progenitor cells (BM-MSCs). The hypothesis that TSPCs possess a mesenchymal stem cell behavior opens a new prospective for tendon regenerative medicine approaches. TSPCs were expanded more rapidly and showed higher plating efficiency when compared with BM-MSCs. Both cell lines expressed identical stem cell markers in vitro and they were able to differentiate towards osteogenic and adipogenic lineages as demonstrated with cytochemical staining and mRNA gene expression. TSPCs showed a positive but limited chondrogenic differentiation compared with BM-MSCs as demonstrated by histological and biochemical analyses. According to our results, equine TSPCs have high clonogenic properties and proliferating potential, they express stem cell markers and have the capability to be multipotent as well as BM-MSCs. These findings suggest that TSPCs may represent a good model for stem cell biology and could be useful for future tendon regenerative medicine investigations.

Size-sieved subpopulations of mesenchymal stem cells from intervascular and perivascular equine umbilical cord matrix.

OBJECTIVES: Umbilical cord matrix (UCM) has been recently proposed as an alternative source of mesenchymal stem cells (MSCs). The aim of this study was to isolate and characterize presumptive stem cells from intervascular and perivascular equine UCM and to obtain homogeneous subpopulations from both sites. MATERIALS AND METHODS: Umbilical cords were processed for retrieval of MSCs. Unsieved cells from intervascular and perivascular portions were evaluated for cell cycle analysis and for immunophenotyping by flow cytometry. Cells from each site were separated into larger and smaller sieved populations using multi-dishes with 8-µm pore transwell inserts. Each cell population was characterized in terms of renewal capability, specific marker expression and differentiation potential. Cryopreservation was performed on sieved cells only. RESULTS: Cells from both areas expressed MSC and pluripotential specific markers and were able to differentiate into mesodermic and ectodermic lineages. The sieving procedure yielded two relatively homogeneous subpopulations with comparable characteristics. Surprisingly, after sieving, large intervascular and small perivascular cells were the most rapidly replicating cells [20.53 and 19.49 cell population doublings (PD) after 31 days respectively] and also showed higher fibroblast colony forming unit frequency. Unsieved cell populations were used as controls, and showed PD of 9.42(intervascular cells) and 8.54 (perivascular cells) after 31 days. CONCLUSIONS: Here, cells from UCM represented an intermediate stage between pluripotent embryonic and adult stem cells. Size-sieving can be used to isolate more rapidly proliferating cell populations.

Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa: a comparative study of amniotic fluid, amnion, and umbilical cord matrix.

The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canine MSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblast-like, and the population doubling time (DT) significantly increased with passage number. For AF- and AM-MSCs, cell viability did not change with passages. In UCM-MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P < 0.05). Amnion and UCM-MSCs expressed embryonic and MSC markers, such as Oct-4 CD44, CD184, and CD29, whereas AF-MSCs expressed Oct-4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLA-DRA1 and DLA-79) were expressed only in AF-MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs.

Fetal adnexa derived stem cells from domestic animal: progress and perspectives.

The fetal adnexa such as umbilical cord, amnion and amniotic fluid have been proposed as ideal sources of different stem cell lineages. Use of adnexal tissue has many potential advantages, including the noninvasive nature of the isolation procedure, the large tissue mass from which cells can be harvested with high efficiency and the potential of these cells to differentiate. Moreover, particularly in human medicine, the harvesting of these tissues is more ethically acceptable making these sources of stem cells very attractive for regenerative therapies and biotechnological applications. The adnexal tissue cells preserve some of the characteristics of the primitive embryonic layers from which they originate. Indeed, many studies indicate that these stem cells exhibit some features of embryonic stem cells as expression of embryonic markers and proliferation capability, without showing immunogenicity. However, the differentiation potential of these cells, either in vivo or in vitro, is intermediate between the pluripotent embryonic stem cells and the multipotent adult stem cells. Non-embryonic extra-fetal derived stem cells have opened new perspectives for developmental biology and for regenerative medicine, not only in humans but also in animals. In this update, we report the state of the art of fetal adnexa-derived stem cells from domestic animals and analyze their applications and potential uses in veterinary medicine.

Morphometric characteristics and chromatin integrity of spermatozoa in three Italian dog breeds.

OBJECTIVES: Studies in many species indicate that variation of spermatozoan head morphology is a sensitive biomarker for abnormal chromatin structure and resultant clinical fertility. This preliminary study evaluated spermatozoan head morphometry in different dog breeds and assessed whether morphometric parameters could reflect spermatozoan DNA fragmentation in dogs. METHODS: Spermatozoan morphometry and DNA quality (measured by TUNEL flow cytometry) were assessed in semen from 11 dogs of three Italian breeds (Cirneco dell'Etna, Piccolo Levriero Italiano and Segugio Maremmano). RESULTS: Morphometric data showed that Segugio dogs had significantly larger (33·67%) spermatozoa and that Piccolo Levrieros had a higher incidence of long (46·75%) and elliptical spermatozoan heads (11·5%) when compared with the samples from other breeds. Moreover, the predominance of elliptical spermatozoa in one dog (23%) was significantly related to the percentage of spermatozoa with fragmented DNA (12·6%), whereas in another dog, where no more than 1% of spermatozoa was elliptical, only 0·36% of spermatozoa had damaged DNA. It is noteworthy that the breeding record of the former dog in the previous 12 months showed poor fertility and fecundity. CLINICAL SIGNIFICANCE: These data suggest that spermatozoan head morphometry could be breed related and that there is a significant correlation between DNA fragmentation and elliptical spermatozoa in individual animals. This finding, albeit limited in our study to a single case, is possibly related to clinical infertility.

Efficacy of tuohy needle in oocytes collection from excised mare ovaries.

TWO METHODS HAVE BEEN DESCRIBED TO RECOVER OOCYTES FROM EQUINE FOLLICLES IN EXCISED OVARIES: aspiration and scraping. Aim of this work was to develop an effective method for collecting equine oocytes using Tuohy needle and comparing this technique to aspiration and scraping, with or without tunica albuginea removal. This hollow hypodermic needle, usually employed for inserting epidural catheters, is designed with a slightly curved tip, shaped similar to a small curette. In unpeeled ovaries, the recovery rates of Tuohy needle group was higher (P < .05) than in the 16 g needle aspiration and in the scraped ovaries (57% versus 36% and 47%) while the rate of cumulus-intact oocytes was higher than aspiration (46.9% versus 39.36%) but lower than scraping (46.97%) (P < .001). In unpeeled ovaries there was significant difference in maturation rate of oocytes recovered by Tuohy needle in respect to peeled ovaries (58.54% versus 50.17%, resp.). Combination of aspiration and scraping by Tuohy needle allows a faster and reliable collection of oocytes suitable for horse IVM.

Time course of in vitro maturation of compact cumulus horse oocytes after roscovitine-induced meiotic inhibition: effects on the coordination between nuclear and cytoplasmic maturation.

This study was carried out to evaluate the usefulness of a pre-maturation step in improving the coordination between cytoplasmic and nuclear maturation of horse compact cumulus oocytes by the addition of roscovitine (ROSC). Oocytes were collected by scraping and pre-cultured for 18 h in a maturation medium TCM199 supplemented with pyruvate, LH, FSH, insulin growth factor (IGF), epidermal growth factor (EGF), insulin, transferrin and selenium (IVM-ROSC) or in a simple medium (M199-ROSC). After pre-maturation, oocytes from both the groups were in part denuded and fixed-stained and in part in vitro matured to assess the kinetic of in vitro maturation (IVM). The nuclear progression and the cytoskeletal organization of microfilaments and cortical granules (CG) of treated and untreated oocytes were assessed by fluorescent probes. Oocytes immediately fixed after recovery and oocytes pre-cultured in M199-ROSC for 18 h did not show metaphase II (MII) plates, whereas in IVM-ROSC group, 6/69 oocytes (8.7%) showed MII plates. After inhibition, during maturation kinetics at 11, 18 and 29 h, maturation rate of M199-ROSC group progressively increased and at 29 h of IVM, reached the maturation rate of control group (13/66, 19.7% vs 31/125, 24.8%). No statistically significant differences in cytoplasmic maturation were found. The number of MII plates after 29 h of IVM, was significantly higher (p < 0.05) in IVM-ROSC group (34/90) compared with M199-ROSC (13/66) and control groups (31/125) as well as the number of oocytes with microfilaments and CG distributed in cortical region (25/34 vs 3/13 and 7/31 respectively). Our results showed that pre-culturing in the presence of Roscovitine in a fully supplemented maturation medium containing gonadotropins and growth factors partially suppressed the meiotic maturation, but established a more suitable environment for improving cytoplasmic maturation of horse compact cumulus oocytes as defined by microfilaments and CG configuration.

Spontaneous lipid peroxidation and sperm metabolism during incubation in media simulating the oviductal microenvironment.

It has been suggested that along the female genital tract spontaneous lipid peroxidation regulates the limit of the lifetime of spermatozoa. We have studied some aspects of rabbit and mouse spermatozoal metabolism during spontaneous lipid peroxidation in the course of the incubation in media which simulate the oviductal environment. The spermatozoa collected at regular intervals after the beginning of incubation were processed for cytochemical detection of cytochrome oxidase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities. Quantitative cytochemical assays were made in situ in individual spermatozoa by microdensitometry. The cytochrome oxidase activity significantly decreased in both species because of damage to mitochondrial enzymes and membranes by radical and non-radical products of lipid peroxidation. The change in lactate dehydrogenase activity indicates that under our experimental conditions the lipid peroxidation process damages membrane permeability more markedly in mouse spermatozoa. The glucose-6-phosphate dehydrogenase activity, which should influence the concentration of reduced glutathione through production of NADPH, is more extensively enhanced in mouse spermatozoa than in rabbit spermatozoa. This is in agreement with the fact that in mouse spermatozoa the glutathione system is the major protective defence against oxidative damage while in rabbit spermatozoa it is superoxide dismutase.

[Cytochemical study of human sperm metabolism during in vitro capacitation after cryopreservation]. / Studio citochimico sul metabolismo degli spermatozoi umani nel corso della capacitazione in vitro anche dopo crioconservazione.

To study the effects of deep freezing on the energy metabolism of human spermatozoa, we investigated, by cytochemical quantitative methods, cytochrome oxidase and lactate dehydrogenase activities of fresh and frozen human spermatozoa during in vitro capacitation. Fresh and frozen human spermatozoa were incubated in Biggers, Whitten and Wittingham's medium supplemented with 15% heat-inactivated human serum. Both histoenzymological reactions can be quantitated and have been evaluated by microdensitometric method. The results indicate that human spermatozoa depend almost entirely on anaerobic glycolysis during in vitro capacitation and suggest that both aerobic and anaerobic metabolism in spermatozoa are only slightly impaired by freezing-thawing and storage.

Microphotometric study of glucose-6-phosphate dehydrogenase activity in epididymal spermatozoa during spontaneous lipid peroxidation.

Mammalian spermatozoa are highly sensitive to lipid peroxidation and the glutathione peroxidase/reductase system provides an effective defense against oxidative damage to different degree in different species. Rabbit spermatozoa rely on superoxide dismutase as the primary enzymatic defense against lipid peroxidation and contain only low detectable endogenous glutathione reductase activity while in mouse spermatozoa the glutathione system is the major protective enzyme against cell damage by autoxidation. We describe a cytochemical quantitative assay for glucose-6-phosphate dehydrogenase activity in rabbit and mouse spermatozoa undergoing spontaneous lipid peroxidation during in vitro incubation. Microdensitometric measurements were made by a Vickers M85 a scanning microdensitometer at lambda 585 nm wavelength. Our findings suggest that in mouse spermatozoa, the enhanced glutathione reductase and peroxidase activities induced by the spontaneous lipid peroxidation increases NADPH production from the pentose phosphate shunt, while in rabbit spermatozoa, NADPH production is much lower.

Effects of deep freezing on the energy metabolism of bovine spermatozoa during in vitro capacitation: A cytochemical approach.

In order to study the effects of deep freezing on the energy metabolism of bovine spermatozoa, a cytochemical quantitative study was carried out by a microdensitometric method on cytochrome oxidase and lactate dehydrogenase (LDH) activities. These were evaluated in situ on individual frozen-thawed bull spermatozoa collected at different times during in vitro capacitation. The results showed that in bull spermatozoa both the initiation of motility and capacity to fertilize eggs were associated with the anaerobic rather than aerobic glycolysis. The freezing-thawing processes and storage in liquid nitrogen induced a general enhancement of both the enzymatic activities examined. The high ionic strength treatment gave rise to a significant but reversible decrease in both the cytochrome oxidase and LDH activities in the fresh as well as in the frozen-stored sperm. The findings, based on cytochemical observations of energy metabolism of spermatozoa and evaluated during in vitro capacitation, suggest that the respiration and the anaerobic glycolysis of spermatozoa seem to be slightly impaired by the freezing-thawing and storage processes.

Cytochemical study on human spermatozoa metabolism during in vitro capacitation.

In eutherian mammalian spermatozoa the capacitation is coupled to a specific type of metabolism, that is glycolysis or oxidative respiration. A cytochemical study was carried out on cytochrome oxidase and lactate dehydrogenase in human spermatozoa collected at different times during in vitro capacitation. Human spermatozoa were incubated in Biggers, Whitten and Wittingham's medium supplemented with 15% heat-inactivated human serum. Both histoenzymological reactions based on oxidative polymerization of diaminobenzidine (cytochrome oxidase) or on tetrazolium salts reduction (lactate dehydrogenase) can be quantitated and have been evaluated by microdensitometric method (Vickers M85). The results suggest that human spermatozoa depend almost quite on the anaerobic glycolysis during in vitro capacitation.