Evaluation of testicular echotexture with Ecotext as a diagnostic method of testicular dysfunction in stallions.

This study aimed to assess if Ecotext, a new software for evaluation of testicular echotexture, is a good method for diagnosis of stallions with testicular dysfunction (TD). Relationships between Ecotext parameters and sperm motility and production, testicular volume, and testicular blood flow were also studied. Ecotext provides a total of six echotexture parameters: Ecotext 1 (black pixels), 2 (white pixels) and 3 (grey pixels), and another 3 parameters related to hypoechogenic areas: Ecotext tubular density (ETD), Ecotext tubular diameter (ETd), and Ecotext tubular area (ETA). Stallions (n = 33) were assessed using proven diagnostic techniques (spermiogram, B-mode and Pulse Doppler ultrasound), and subsequent analysis with Ecotext. Animals were classified as "control stallions" (n:21, acceptable semen quality), and "stallions with TD" (n:12, poor semen quality (TM < 60%, PM < 45% and total nº of sperm with PM < 2000 × 106 spz), that were subdivided into "induced TD group" (immunized, anti-GnRH vaccine) and "acquired TD group". The acquired TD group showed differences in all Ecotext parameters in relation to controls (Ecotext 1:0.11 ± 0.17 vs 2.82 ± 2.52, Ecotext 2:1584.0 ± 575.8 vs 388 ± 368.2, Ecotext 3:134.2 ± 9.26; ETA: 2.14 ± 0.59 vs 5.40 ± 1.90; ETd: 65.66 ± 6.27 vs 86.93 ± 10.65 and ETD: 92.35 ± 11.24 vs 132.10 ± 16.35, p ≤ 0.001). Results suggest acquired TD stallions were suffering testicular degeneration with loss of architecture and function as all Ecotext parameters were altered in relation to controls. Induced TD horses only showed a reduction in ETD (116.2 ± 8.59 vs 132.10 ± 16.35, p ≤ 0.001), despite all sperm parameters being worse. These findings suggested immunized stallions probably only experience an acute loss of testicular functionality and parenchyma architecture is likely not affected since differences in Ecotext parameters with control stallions were not detected. ETD was the best parameter to identify animals with TD (AUC: 0.84, optimal cut-off value of 124.3 seminiferous tubules/cm2). Correlations were found between ETD and Doppler indices (PI: 0.60; RI: 0.47 p ≤ 0.001), total testicular volume (r: 0.48; p ≤ 0.05) and sperm motility (TM:0.51; and PM:0.54; p ≤ 0.001) and production (r:0.51; p ≤ 0.001). In summary, Ecotext could identify changes in testicular echotexture of stallions with TD. Results open the possibility for new research focused on establishing the relationship between Ecotext parameters and histomorphometry features in stallion testes.

Identification of sperm head proteins involved in zona pellucida binding.

STUDY QUESTION: Which human sperm proteins interact with zona pellucida (ZP) glycoproteins, ZPA/2, ZPB/4 and ZPC/3? SUMMARY ANSWER: Co-precipitation experiments with recombinant human ZP (rhZP) coated beads demonstrated interactions with various proteins, including glutathione S-transferase M3 (GSTM) with ZPB/4 and voltage-dependent anion channel 2 (VDAC2) with ZPA/2 and ZPC/3. WHAT IS KNOWN ALREADY: Regarding sperm-ZP binding, several target spot/proteins have been detected in several species, but not all have been characterized. The limit of these studies was that a mixture of the different ZP glycoproteins was used and did not allow the identification of the specific ZP glycoprotein (ZPA/2, ZPC/3 or ZPB/4) involved in the interaction with the sperm proteins. STUDY DESIGN, SIZE, DURATION: To identify the human sperm proteins interacting with the oocyte ZP, we combined two approaches: immunoblot of human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far western blot of human sperm proteins overlayd by each of the rhZP proteins. MATERIALS, SETTING, METHODS: We used rhZP expressed in Chinese hamster ovary (CHO) cells and ASA eluted from infertile patients undergoing IVF failure. Sperm proteins separated by two-dimensional (2D) electrophoresis recognized by both sperm-eluted ASAs from infertile patients and rhZP were identified by mass spectrometry (MALDI-MS/MS). Some of these proteins were further validated by co-precipitation experiments with rhZP and functional zona binding tests. MAIN RESULTS AND THE ROLE OF CHANCE: We identified proteins that are glycolytic enzymes such as pyruvate kinase 3, enolase 1, glyceraldehyde-3-phosphate dehydrogenase, aldolase A, triosephosphate isomerase, detoxification enzymes such as GSTM or phospholipid hydroperoxide glutathione peroxidase, ion channels such as VDAC2 and structural proteins such as outer dense fibre 2. Several of the proteins were localized on the sperm head. However, these proteins have also been described to exert other functions in the flagellum. Co-precipitation experiments with rhZP-coated beads confirmed the direct interaction of GSTM with ZP4 and of VDAC2 with ZP2 and ZP3. LIMITATIONS, REASONS FOR CAUTION: We used recombinant ZP in place of native ZP. Thus, the post-translational modifications of the proteins, such as glycosylations, can be different and can influence their function. However, CHO cell-expressed rhZP are functional, e.g. can bind human spermatozoa and induce the acrosome reaction. Moreover, the identification of relevant proteins was limited by the need for sufficient amounts of proteins on the preparative 2D-gel to be subsequently analysed in MALDI-TOF MS/MS. WIDER IMPLICATIONS OF THE FINDINGS: Our results bring new insights on the ability of sperm proteins to exert several functions depending on their sub-cellular localization, either the head or flagellum. Their multiple roles suggest that these sperm proteins are multifaceted or moonlighting proteins. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the grant ReproRio (CNRS, INRA, INSERM and CEA) and the Société d'Andrologie de Langue Française. TRIAL REGISTRATION NUMBER: Not applicable.

Colloidal centrifugation of stallion semen results in a reduced rate of sperm DNA fragmentation.

Stallion spermatozoa recovered and examined immediately after colloidal centrifugation resulted in a higher straight-line velocity (VSL) than sperm processed using direct conventional centrifugation (p = 0.000), but there was no differences in the progressive motility or sperm DNA fragmentation (SDF) as determined by the sperm chromatin dispersion assay. However, when centrifuged spermatozoa were incubated at 37 °C for 24 h to determine the rate of SDF (r-SDF), a lower r-SDF (p = 0.0011) was observed in those sperm recovered after colloidal separation (0.5 ± 0.1%/h) compared to direct (1.2 ± 0.4%/h) or no centrifugation (r-SDF = 1.2 ± 0.3%/h). These results confirm that colloidal separation of stallion spermatozoa results in prolonged sperm DNA longevity, but these differences were only apparent following a period of incubation and dynamic assessment. Consequently, we strongly recommend the use of the dynamic form of the SDF assay for evaluating centrifugation and/or other ex vivo procedures, as a single basal assessment of SDF may inadvertently result in a false-positive evaluation of DNA quality.

Simple and economic colloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure.

For many years in human assisted-reproduction procedures there have been special protocols to prepare and improve sperm quality. Colloidal centrifugation (CC) is a useful technique that has been proved to enhance semen quality by selection of the best spermatozoa for different species. Its use is recommended to improve fertility of subfertile stallions but current CC protocols are clinically complicated in the equine sperm processing technique due to economic and technical difficulties. The aim of this study was to determine the optimal processing procedures to adapt the use of a CC product (EquiPure™) in the equine reproduction industry. A total of nineteen ejaculates were collected from 10 Purebred Spanish Horses (P.R.E horses) using a Missouri artificial vagina. Gel-free semen aliquots were analyzed prior to treatment (control). Semen was subjected to one of six CC protocols with EquiPure™ and centrifuged samples were statistically evaluated by ANOVA and Duncan tests (p<0.05) for sperm quality and recovery rate. We obtained higher values by colloidal centrifugation in LIN, STR and BCF variables and DNA fragmentation index trended to be lower in most of the CC protocols. The studied protocols were shown to be as efficient in improving equine sperm quality as the current commercial EquiPure™, with the added advantage of being much more economical and simple to use. According to these results it seems to be possible to incorporate single layer and or high colloidal centrifugation volume protocols what would make them simple, economic and clinically viable for the equine sperm processing procedure.

[Role of the female environment in sperm capacitation]. / La capacitation in vivo: influence de l'environnement féminin.

The female genital tract plays a vital role in ensuring successful fertilization and normal early embryonic development. The process of capacitation needs an active and specific coordination between the female tract, ovulation and the spermatozoa. During sperm ascension towards the fertilisation site, a final maturation of male gametes, also called sperm capacitation, takes place in a well-controlled manner. Before ovulation, most of spermatozoa bind oviduct, in the isthmic region that plays a role of sperm reservoir. During sperm binding to the epithelial cells the capacitation process is delayed and spermatozoa can survey several days. The capacitation is completed when spermatozoa are released at the ovulation time. They are then guided towards the oocyte likely thanks to combination of female factors, including those provided from oviduct secretions and cumulus cells.

Mouse models for identifying genes modulating fertility parameters.

Fertility can be defined as the natural capability of giving life. It is an important factor both for human medicine, where ~10% of the couples call for the services of assisted reproductive technologies, and for species of economic interest. In particular, in dairy cows, the recent years have seen a kind of competition between milk production and fertility, and genes improving fertility are now considered as parameters to be selected for. The study of fertility pathways is nevertheless made difficult by the strong impact of environmental factors on this parameter, as well as by the number of genes potentially involved (as shown by systematic transcriptome analysis studies in the recent years). One additional level of complexity is given by the fact that factors modulating fertility will probably be sex specific. The usage of mouse models has been one of the solutions exploited for tackling with these difficulties. Here, we review three different approaches using mice for identifying genes modulating fertility in mammals: gene invalidation, positional cloning and in vitro mutagenesis. These three approaches exploit specific characteristics of the mouse, such as the possibility of controlling precisely the environment, an excellent genetic characterization and the existence of genomic and molecular tools equalled only in humans. Many indications suggest that at least some of the results obtained in mice could be easily transposed to the species of interest.

Pharmacokinetics of a single intravenous dose of marbofloxacin in adult donkeys.

Six donkeys each received 2 mg/kg marbofloxacin as a 10 per cent aqueous solution administered intravenously. Principal pharmacokinetic parameters were determined and two efficacy indices were computed by using pharmacokinetic parameters and selected mic90 values of marbofloxacin against pathogenic equine strains to predict the efficacy of the drug at this dose. The pharmacokinetics of marbofloxacin in donkeys was characterised by a large mean volume of distribution at a steady state (1.15 [0.09] l/kg) and a long mean (sd) elimination half-life of 9.24 (1.96) hours. It was also characterised by a relatively slow total body clearance of 0.10 (0.02) l/kg/hour, slower than in horses. Using mic90 values of marbofloxacin against pathogenic equine strains with a daily dose of 2 mg/kg, appropriate values of efficacy indicators were obtained only for Enterobacteriaceae. Daily intravenous doses of 0.33, 2.62 and 20 mg/kg were calculated for evaluation in clinical trials of infections due to Enterobacteriaceae, Staphylococcus aureus and Streptococci, respectively.

Spatio-developmental distribution of the prion-like protein doppel in Mammalian testis: a comparative analysis focusing on its presence in the acrosome of spermatids.

The first prion-like protein doppel, officially designed as prion protein dublet, does not seem to be needed for prion disease progression, whereas its physiological function seems to be related to male fertility. Its expression is primarily detected in the male genital tract, and Prnd-inactivated male mice are sterile. We investigated the location of Doppel in the testis of various species of mammal to determine its physiological function. Doppel is expressed early during ontogenesis, and is found in both germ cells and Sertoli cells in mice, rats, boars, and humans. Doppel is permanently expressed in the Sertoli cells but at different levels according to species. Its expression in testicular germ cells was primarily detected in spermatids, with a transient presence in the acrosome. These data suggest that Doppel may play a physiological role in acrosome biogenesis and may be of use in studies of patients suffering from idiopathic infertility.

Efecto de la concentración del Glicerol y su posterior eliminación en la criopreservación de semen de asno zamorano-leonés / Effect of Glycerol concentration and subsequent elimination in the criopreservation off zamorano-leonéz donkey

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Progesterone induces hyperpolarization after a transient depolarization phase in human spermatozoa.

Progesterone (P4) induces a membrane depolarization and various ion fluxes (chloride efflux, sodium and calcium influxes), which are required for the human sperm acrosome reaction (AR). By use of the potentiometric fluorescent dye DiSC3(5) and two different technical approaches, the present study aimed to quantify and further analyze P4-induced modifications in membrane potential in capacitated human spermatozoa. Spectrofluorimetric analysis revealed that the mean resting membrane potential of sperm was -58 +/- 2 mV (n = 12). When 10 microM P4 was added, the sperm membrane depolarized by approximately +15 mV, partly driven by a Cl- efflux. It subsequently repolarized to reach a significant lower potential than the initial resting potential in two thirds of the tested samples. The flow cytometry analysis showed a heterogeneous resting membrane potential and revealed that the depolarization-hyperpolarization events concerned only subpopulations, between 3% and 40% of the sperm cells according to the samples (n = 7). We hypothesize that P4 has a beneficial effect on the ability of zona pellucida to promote the AR in a sperm subpopulation by increasing the number of hyperpolarized cells presenting a membrane potential that is compatible with the opening of T-type calcium channels by subsequent zona pellucida-induced depolarization.

The acrosome reaction in human spermatozoa.

During gamete interaction, sperm acrosome reaction (AR) induced by oocyte investment is a prerequisite event for the spermatozoa to pass through the zona pellucida (ZP), fuse with and penetrate the oocyte. Progesterone (P4), secreted by cumulus cells, is an important cofactor for the occurrence of this exocytosis event. The AR results from the fusion between outer acrosomal and plasma membranes, leading to inner acrosomal membrane exposure. Binding of agonists, P4 or ZP3 glycoprotein, to plasma membrane sperm receptors activates intraspermatic signals and enzymatic pathways involved in the AR. Among the proteins or glycoproteins described as potential sperm receptors for ZP, Gi/Go protein-coupled and tyrosine kinase receptors have been described. Sperm receptors for P4 are poorly characterized, except a putative GABA(A)-like receptor. ZP- and P4-promoted AR is mediated by an obligatory intracellular calcium increase, appearing first at the acrosome equatorial segment and spreading throughout the head. The plasma membrane channels involved in calcium entry are operated by a plasma membrane depolarization and protein phosphorylations mediated by protein kinase C and tyrosine kinase protein. Part of the calcium increase could also be due to intracellular store release through IP3- and nucleotide (cAMP)-gated channels. Besides adenylate cyclase and phospholipase C activations, intracellular calcium increase also stimulates PLA2 activity and actin depolymerization, leading to membrane fusion. Evaluation of AR by staining or fluorescent probes can be useful to predict fertilization success and to direct the therapeutic strategy in male infertility.

Induction of a sodium ion influx by progesterone in human spermatozoa.

In human spermatozoa, progesterone (P(4)) induces a depolarization of the plasma membrane, a rapid calcium (Ca(2+)) influx, and a chloride efflux. The sodium ion (Na(+)) was partly responsible for the P(4)-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P(4) induced a Na(+) influx and whether voltage-operated channels were involved in Na(+) and/or Ca(2+) entries. We found that 10 microM P(4) significantly increased intracellular Na(+) concentration from 17.8 +/- 2.0 mM to 27.2 +/- 1. 6 mM (P < 0.001). Prior incubation of spermatozoa with 10 microM flunarizine, a Na(+) and Ca(2+) voltage-dependent channel blocker, inhibited the sodium influx induced by 10 microM P(4) by 84.6 +/- 15.4%. The Ca(2+) influx induced by 10 microM P(4) was also significantly inhibited in a Na(+)-containing medium by 10 microM flunarizine or 10 microM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca(2+) influx induced by 10 microM P(4) in spermatozoa incubated in Na(+)-depleted medium. The P(4)-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na(+)-containing medium as compared to Na(+)-depleted medium. These data demonstrate that P(4) stimulates a Na(+) influx that could be involved in the AR completion. They also suggest that voltage-dependent Na(+) and Ca(2+) channels are implicated in P(4)-mediated signaling pathway in human spermatozoa.

[The movement of the human spermatozoon]. / Le mouvement du spermatozoïde humain.

The human sperm flagellum is composed by an axoneme made up of peripheral doublets of longitudinal microtubules on which are fixed dynein arms and radial spokes. A mechanochemical cycle of attachment-detachment of dynein arms on the adjacent microtubules results in a sliding/bending or microtubules. This process repeated along the flagellum, induces the propagation of a wave. The progressive movement of ejaculated spermatozoa is modified during their transport through the feminal genital tract under the influence of microenvironmental factors. The microfibrillar structure of cervical mucus constraints the flagellar wave amplitude whereas the tubal or follicular secretions induce an hyperactivation of the movement characterized by a loss of progression and a high degree of flagellar curvature. Sperm movement modifications are depending on external regulatory factors among which some could be secreted by the oocyte or by the perioocyte layers to create a sperm chemotaxis which would optimize the gametic interaction. The external factors modulate sperm movement through their interaction with membrane "receptors" and intracellular messengers such as cyclic AMP, ATP, Calcium, pH. These latter control dynein-microtubules interaction through a phosphorylation-dephosphorylation process of axonemal proteins.

Use of the single-cell gel electrophoresis assay for the immunofluorescent detection of specific DNA damage.

The single-cell gel electrophoresis assay or comet assay is now a widely used method to assess the level of DNA damage in irradiated or chemically modified cells. We propose an adaptation of the currently applied protocol, aimed at singling out a defined modified base, using an immunodetection approach. After the electrophoresis step, the DNA tail moment was measured using ethidium bromide. Simultaneously, cyclobutane pyrimidine dimers (CPDs), the targeted lesions, were revealed by an indirect immunofluorescence detection using a specific monoclonal antibody. The assay was validated on human fibroblasts exposed to UVB light. The dose-response curves were established, showing a linear increase of the antibody response with the dose between 1000 and 10,000 J/m2. The detection limit of the method was 500 J/m2. Digestion of the CPDs, induced at 3000 J/m2, with T4 endonuclease V led to a marked decrease of the antibody response, confirming the specificity of the assay. A preliminary repair experiment is reported in which the tail moment of the comets together with the antibody response are measured, showing the disappearance of 80% of the antibody fixation sites within 48 h.

The reactivation of demembranated human spermatozoa lacking outer dynein arms is independent of pH.

The effect of pH, Mg-ATP, and free calcium on activity of the inner dynein arm was investigated using demembranated human spermatozoa lacking the outer dynein arms (LODA). The results were compared with those obtained for demembranated-reactivated normal spermatozoa to evaluate the functional properties of the inner and outer dynein arms in axonemal motility. The reactivation of Triton X-100-demembranated LODA spermatozoa was analysed at various pHs and concentrations of Mg-ATP and calcium using video recordings. The percentage of reactivated LODA spermatozoa as a function of Mg-ATP concentration was not dependent on pH, whereas reactivation of normal human spermatozoa is pH dependent. This suggests that there may be a pH-dependent regulatory mechanism associated with the outer dynein arms. A delay in the principal bend propagation of normal and LODA reactivated cells was found at pH 7.1. This disappeared at pH 7.8 in normal but not in LODA populations. This suggests a role for outer dynein arms in the initiation of the propagation of flagellar bends at alkaline pH. The level of LODA and normal sperm reactivation both depended on the calcium concentration in the medium. At lower free calcium concentrations, the reactivation level and beat frequency of reactivated cells were higher. Our results suggest a functional difference between outer and inner dynein arms of human spermatozoa based on a differential pH sensitivity. Moreover, calcium seems to exert its regulatory action elsewhere than on the outer dynein arms.

RU486 inhibits penetration of human spermatozoa into zona-free hamster oocytes.

Previous studies have shown that RU486 decreases the concentration of intracellular calcium ([Ca2+]i) in human spermatozoa in vitro and partially antagonizes the effect of progesterone on calcium influx and sperm acrosome reaction. The present study has examined the effect of RU486 on the penetration of human spermatozoa into zona-free hamster oocytes. RU486 (10 microM) decreased significantly the rate of penetration of zona-free hamster oocytes by human spermatozoa, and nearly abolished penetration when present in the medium at concentrations of 50 and 100 microM. RU486 must be present in the medium to exert its inhibitory effect. At the same concentration (10 microM) as RU486, progesterone was unable to reverse the inhibitory effect of RU486 on the penetration rate of human spermatozoa.

Protein-energy malnutrition during gestation and lactation in rats affects growth rate, brain development and essential fatty acid metabolism.

The influence of feeding a low protein diet to rat dams during gestation and lactation on lipid metabolism in pups was studied. Wistar rats were fed 5, 10, 15 and 25% dietary protein during gestation and lactation. Pup growth was monitored until weaning, and brain weight, protein concentration, proteolipid concentration and total lipid phosphorus concentration of brain were analyzed. The levels of fatty acids in dam milk as well as in pup liver phospholipids and brain prosphatidylcholine and phosphatidylethanolamine were determined. The progressive deprivation of maternal dietary protein produced a reduction in the total saturated fatty acid concentration of dam milk and an increment in the concentration of nonmetabolized linoleic acid. Pup body and brain weights as well as proteolipid, protein and total lipid phosphorus concentrations in brain were reduced in proportion to the degree of dietary protein deficiency. The products:precursor ratio of (n-6) fatty acids in liver phospholipids revealed an impairment in the elongation-desaturation pathway due to maternal protein deficiency. Both (n-6) and (n-3) polyunsaturated fatty acids within brain phosphatidylethanolamine were decreased by reduced maternal dietary protein intake, whereas only the linoleic acid-derived products were similarly affected in the corresponding phosphatidylcholine fraction. These results demonstrate the widespread and profound deleterious effects of low protein levels of maternal diet on the growth rate, brain development and fatty acid metabolism in rat pups.

RU486 and calcium fluxes in human spermatozoa.

RU486 decreases the intracellular free calcium concentration in human spermatozoa incubated in capacitation medium. We investigated the calcium fluxes that this progesterone antagonist acts on. We found that 10(-5) M RU486 slowed down the basal calcium influx while progesterone accelerated it. Moreover, in the same way as RU486 inhibits the calcium increase promoted by progesterone, it inhibited the calcium influx stimulated by 10(-6) M thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor. So we speculated that RU486 does not necessarily compete for the same binding sites as progesterone to exert its inhibitory action. In the absence of extracellular calcium, pretreatment by 10(-5) M RU486 decreased the peak of [Ca2+]i released by 5 microM ionomycin. This indicates that RU486 could also act at the membrane level of some intracellular organelles of calcium storage.

[Sperm motility and fertilization]. / Mouvement des spermatozoïdes et fécondation.

Sperm motility is a key function in the process of fertilization. Significant gaps in the knowledge of the mechanisms of control and modulation of sperm motility still remain. It is well known that the percentage of motile sperm is correlated with fertility in vivo but the recent development of assisted reproductive technologies and the extensive use of objective methods to assess sperm movement in vitro indicate that the quality of sperm movement is important too for fertilization. However, the exact pattern of motility characteristics to fulfill fertilization in vitro and in vivo are not clearly established essentially because of the lack of multiparametric and controlled studies in vitro and prospective studies in vivo. There is also presently no evidence for an improvement of fertility when using different pharmacological approaches to increase sperm motility in vitro. Therefore, further efforts are required to improve our basic knowledge of the relations between sperm motility and fertility with the aim to adapt and apply it to the problem of male infertility.

[Effect of the administration of corn oil on the composition of fatty acids of plasma phospholipids in normal and malnourished infants fed with cow's milk]. / Efecto de la administración de aceite de maíz sobre la composición en ácidos grasos de los fosfolípidos del plasma de lactantes normales y desnutridos alimentados con leche de vaca.

The effect of corn oil diet administration on the essential fatty acids (EFAs) profiles was evaluated in plasma phospholipids from normal and malnourished cow's milk fed infants nursing infants. A control group of only breast-fed was also selected for this study. The fatty acid composition was determined by gas-liquid chromatography and used as biochemical variable for evaluating EFA status. A fall in the proportion of fatty acids concomitant with an increase in the saturated fatty acids, consistent with a pattern of essential fatty acid deficiency (EFAD) was observed in the cow's milk fed infants, either normal or malnourished (Table 2). The corn oil administration was capable of restoring the fatty acid profile to normal values, similar to the values of the control group of breast-fed infants, even in malnourished infants, although during the 15 days test they did not correct their clinical syndrome of malnutrition (Table 3). Calculation of the product-precursor of the linoleic acid provided evidence for the positive effect of the corn oil administration.