Short communication: determination of lactoferrin in Feta cheese whey with reversed-phase high-performance liquid chromatography.

In the current paper, a method is introduced to determine lactoferrin in sweet whey using reversed-phase HPLC without any pretreatment of the samples or use of a separation technique. As a starting point, the most common HPLC protocols for acid whey, which included pretreatment of the whey along with a sodium dodecyl sulfate-PAGE step, were tested. By skipping the pretreatment and the separation steps while altering the gradient profile, different chromatographs were obtained that proved to be equally efficient to determine lactoferrin. For this novel 1-step reversed-phase HPLC method, repeatability was very high over a wide range of concentrations (1.88% intraday to 5.89% interday). The limit of detection was 35.46µg/mL [signal:noise ratio (S/N)=3], whereas the limit of quantification was 50.86µg/mL (S/N=10). Omitting the pretreatment step caused a degradation of the column's lifetime (to approximately 2,000 samples). As a result, the lactoferrin elution time changed, but neither the accuracy nor the separation ability of the method was significantly influenced. We observed that this degradation could be easily avoided or detained by centrifuging the samples to remove fat or by extensive cleaning of the column after every 5 samples.

Bowel intussusception in adults: a report of three interesting cases and current trends for diagnosis and surgical management.

BACKGROUND: Bowel intussusception in adults remains a rare and constant diagnostic challenge for surgeons. It has an incidence of around 2-3 new cases per million per year, and its primary cause is benign or malignant neoplasms of the small bowel and colon. This report aims to outline the importance of high clinical suspicion regarding intussusception in adults presenting with abdominal pain in the emergency department.  Case report: This is a retrospective review of three cases of adult ileocecal intussusception that were treated in a single surgical department in three years (2015-2018). All patients underwent right hemicolectomy in keeping with the principles of surgical oncology. Each patient had a different clinical presentation, while, in terms of the underlining pathology, the first had an adenocarcinoma of the ascending colon, the second an adenocarcinoma of the ileocecal valve, and the third one an inflammatory fibroid polyp of the ileocecal valve, also known as Vanek's tumor. CONCLUSION: Large bowel intussusception in adults is quite an interesting entity, not only for its rarity but for its non-specific and atypical clinical presentation as well. High suspicion from the clinician's part and availability of a computed tomography scan is the key to diagnosis. It is not unusual for imaging modalities to be unable to identify the cause of the intussusception. Thus, surgery is always the preferred method of treatment, as, more often than not, a neoplasm of the small or the large bowel is the underlining pathology.  HIPPOKRATIA 2019, 23(1): 37-41.

Grape pomace improves performance, antioxidant status, fecal microbiota and meat quality of piglets.

In the present study, grape pomace (GP) was used as feed additive in the diet of weaned piglets in order to develop innovative feedstuffs and to investigate their potential beneficial effects on welfare, productivity and meat quality. For examining the antioxidant capacity of the experimental feeds, 24 piglets of 20 days old were assigned to two experimental groups receiving standard or experimental diet for 30 days. Blood and tissues collections were performed at four different time-points, 2, 20, 35 and 50 days post birth. The collected tissues were brain, heart, kidney, liver, lung, quadriceps muscle, pancreas, spleen and stomach. The following oxidative stress markers were assessed: reduced glutathione (GSH), catalase activity, total antioxidant capacity (TAC), thiobarbituric acid reactive substances (TBARS), protein carbonyls (CARB) and H2O2 decomposition activity. The effect on bacterial growth was assessed by examining microbial populations in piglets' fecal microbiota. Furthermore, the average daily gain (ADG) was calculated and the fatty acid profile of quadriceps muscle was assessed. The results showed that piglets fed with the diet supplemented with GP, had significantly increased antioxidants mechanisms in almost all the tissues as shown by increases in GSH, H2O2 decomposition activity and TAC compared with control group. Piglets fed with the experimental diet exhibited decreased oxidative stress-induced damage to lipids and proteins as shown by decreases in TBARS and CARB in GP group compared with control. In addition, the experimental diet increased significantly ADG (by 23.65%) (P<0.05) and enhanced the growth of facultative probiotic bacteria (by up to 1.2 log colony forming units (CFU)/g) (P<0.05) and lactic acid bacteria (by up to 2.0 log CFU/g) (P<0.05) in GP group compared with the control group. GP supplementation inhibited the growth of pathogen populations such as Enterobacteriacae (by up to 1.8 log CFU/g) (P<0.05) and Campylobacter jejuni (by up to 1.0 log CFU/g) (P<0.05). Regarding fatty acid composition of meat, GP inclusion in piglets' diet increased significantly n-3 fatty acids (EPA; C20 : 5n-3, DHA; C22 : 6n-3, α-linolenic acid; C18 : 3n-3) and decreased significantly n-6/n-3 ratio compared with control (P<0.05). The results suggested that dietary GP supplementation may have a beneficial impact on piglets' welfare and may improve productivity as well as meat quality.

Oocyte pick-up in juvenile lambs affects neither onset of puberty nor their future fertility.

A study was designed to evaluate whether the time of onset of puberty and fertility of young ewe lambs would be affected by oocyte pick-up conducted in single or repeated sessions during the first months of lambs' live. Five groups of lambs from the Karagouniko breed were used (A-E each n=12). In group A no treatments were applied (control group) while, laparoscopical follicular aspiration (OPU) was performed early in the third, fourth and fifth month of lambs age (groups C-E, respectively). From the second to fifth month of their age, group B lambs were aspirated four times in monthly intervals. All lambs were weighed at birth, weaning, at second month and monthly thereafter until the eighth month of age. Progesterone priming and ovarian stimulation by serial FSH administrations proceeded each OPU session. To determine onset of puberty blood progesterone concentration was assayed in samples collected initially every week and after the seventh month of age twice weekly. From the seventh month a fertile ram was introduced in each group and oestrous behavior/mating was daily monitored and recorded. Pregnancy diagnosis was carried out by transabdominal ultrasound scanning 55 days after rams' removal. At the fourth and fifth month of age group B lambs were lighter (p<0.05) than controls, but this difference was later equalized. The time of onset of puberty did not differ between groups (p=0.069) and ranged between 224 and 270 days. Some animals (n=15) entered puberty with a full-length luteal phase having progesterone concentration greater than 1ng/ml, while others (n=32) exhibited one or two short luteal phases before luteal length restoration. During the first breeding season 41 animals were fertilized and maintained pregnancy to term, without noticeable differences between groups (p=0.555). During the second breeding season, all ewes were naturally served and lambed at the expected time. It is concluded that OPU in young dairy lambs does not affect the time of onset of puberty, the endocrine profile of the lambs and it does not compromise their future fertility even if it is applied at four successive months.

Purification and properties of carnitine dehydrogenase from Pseudomonas putida.

Carnitine dehydrogenase (carnitine:NAD+ oxidoreductase, EC 1.1.1.108) from Pseudomonas putida IFP 206 catalyzes the oxidation of L-carnitine to 3-dehydrocarnitine. The enzyme was purified 72-fold to homogeneity as judged by polyacrylamide gel electrophoresis. The molecular mass of this enzyme is 62 kDa and consists of two identical subunits. The isoelectric point was found to be 4.7. the carnitine dehydrogenase is specific for L-carnitine and NAD+. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0 and 7.0 in the reduction reaction. The optimal temperature is 30 degrees C. The Km values for substrates were determined.

Luteal stage dependence of pituitary response to gonadotrophin-releasing hormone in cyclic dairy ewes subjected to synchronisation of ovulation.

Possible hormonal aberrations precluding conception or maintenance of pregnancy in dairy ewes subjected to ovulation synchronisation were investigated in this study. The pituitary response to exogenous gonadotrophin-releasing hormone (GnRH) was tested at different luteal stages in 36 ewes. Oestruses were synchronised by using progestagen-impregnated sponges and the animals were randomly allotted into one of three treatment groups (A, B and C; n = 12 for each). Treatments commenced on Days 4, 9 and 14 of the new cycle (oestrus was defined as Day 0). Ewes were given two GnRH injections, 5 days before and 36 h after a prostaglandin F2+/- (PGF2+/-) injection, and the animals were inseminated 12-14 h after the second GnRH injection (modified OVSYNCH). For luteinising hormone (LH) determination blood samples were withdrawn from six ewes of each group at the time of GnRH administration, and 30, 90, 180, 270 and 360 min later. Progesterone was assayed in samples taken every other day starting from oestrus and for 17 days after the second GnRH injection, and in an additional sample collected on the day of insemination. After the first GnRH injection, the LH concentration was higher in Group C than in Groups B and A (mean +/- s.d.: 64.8 +/- 10.0 ng mL(-1), 41.3 +/- 3.7 ng mL(-1) and 24.6 +/- 9.0 ng mL(-1), respectively; P < 0.05), whereas after the second GnRH injection a uniform LH release was found in all groups. PGF2+/- caused a significant decrease in progesterone (P4) concentration in all groups; however, at artificial insemination ewes that conceived had significantly lower P4 concentration in comparison with those that failed to conceive. As early as Day 5, pregnant animals had higher P4 concentrations than non-pregnant animals. Overall, 21 animals conceived (seven, nine and five ewes from Groups A, B and C, respectively). These results indicate that the proposed protocol is equally effective in inducing a preovulatory LH surge at any stage of the luteal phase, and that elevated P4 concentration along with a delayed P4 increase should be considered as a causative factor for inability to conceive.

3-Chloroacetylpyridine adenine dinucleotide phosphate, an alkylating analogue of NADP+.

An alkylating analogue of NADP+ the 3-chloroacetylpyridine adenine dinucleotide phosphate was prepared from 3-diazoacetylpyridine adenine dinucleotide phosphate which was obtained by enzymatic transglucosidation of NADP+. The 3-diazoacetylpyridine adenine dinucleotide phosphate proved to be more unstable when compared to the corresponding NAD+ analogue. The alkylation of several dehydrogenases using this alkylating analogue is mentioned.

Purification, characterisation, and gene cloning of transglutaminase from Streptoverticillium cinnamoneum CBS 683.68.

The transglutaminase (TGase; EC 2.3.2.13) from Streptoverticillium cinnamoneum CBS 683.68 has been purified, characterised and its gene cloned. The purified enzyme had a relative molecular mass of 37,660 determined by mass spectrometry and contained a single Cys residue that was essential for the catalytic activity. Contrary to eukaryotic TGases, this enzyme was calcium-independent. The fact that TGase was capable to incorporate a wide variety of aliphatic and aromatic non-polar compounds suggested that the amine fixation site could be an hydrophobic pocket. S cinnamoneum CBS 683.68 TGase was synthesised as a protein precursor of 411 amino acid residues corresponding to a signal peptide of 81 amino acid residues and a mature TGase of 330 amino acid residues. Amino acid sequence analysis revealed that the S cinnamoneum CBS 683.68 TGase had little sequence homology with eukaryotic TGases, but shared high identity with the sequence of Streptoverticillium strain S-8112. In accordance with kinetics data, hydropathy analysis showed that the active site of the enzyme was in an hydrophobic environment as for eukaryotic TGases.

Rapid and specific identification of nitrile hydratase (NHase)-encoding genes in soil samples by polymerase chain reaction.

A polymerase chain reaction (PCR) protocol was developed for the specific detection of genes coding nitrile hydratase (NHase). Primer design was based on the highly conserved sequences found in the coding region of the alpha-subunit gene corresponding to the metal-binding site. Purified genomic DNA from bacterial strains or directly from soil can serve as the target for the PCR, thus affording a simple and rapid method for screening NHase genes. The primer pairs, NHCo1/NHCo2 and NHFe1/NHFe2 yield PCR products corresponding to a partial coding sequence of cobalt and iron NHase genes, respectively. Using the PCR method, both types of iron- and cobalt-NHase-encoding genes were detected in DNA from pure cultures and soil samples. Furthermore consensus primers allowed rapid cloning and expression of novel NHases in Escherichia coli.

The presence of environmental pollutants in the follicular fluid of farm animals (cattle, sheep, goats, and pigs).

Organochlorine pesticides and polychlorinated biphenyls are widely used in agriculture and industry, respectively. The present study assessed the burden of environmental pollutants in the follicular fluid of farm animals (cattle, sheep, goats, and pigs). An analytical method combining a solid-phase extraction with (C(18)) for clean-up and GC-electron capture detection using a capillary column was implemented for isolation and determination of organochlorine pesticides and polychlorinated biphenyls (PCBs). Of the organochlorine pesticides, hexachlorobenzene (HCB), hexachlorocyclohexane (HCH, alpha-, beta-, and gamma-isomers), dieldrin, heptachlor epoxide, and the DDT-related chemicals (o,p'-DDE, p,p'-DDE, p,p'-DDD, p,p'-DDT) were detected and of the PCBs, the congeners PCB-52, -101, -138, -153, and -180 were detected. In all species of farm animals, the most frequently detected pollutant was gamma-HCH (90-100% of samples) followed by HCB (80-100%), and p,p'-DDE (75-90.91%). Species differences in the concentrations of HCB, beta-HCH, heptachlor epoxide, and DDT-related chemicals in follicular fluid were noted as well as differences in the concentrations of some pollutants within the same species.

The presence of environmental pollutants in the semen of farm animals (bull, ram, goat, and boar).

Organochlorine pesticides and polychlorinated biphenyls are widely used in agriculture and industry, respectively, and may affect male reproduction function. Although several pollutants have been detected in human semen, similar studies in farm animals have not appeared. In the present study, the semen of bulls, rams, goats, and boars was assayed for the organochlorine pesticides hexachlorobenzene (HCB), hexachlorocyclohexane (HCH, alpha-, beta-, and gamma-isomers), dieldrin, and heptachlor epoxide, for DDT-related chemicals (o,p'-DDE, p,p'-DDE, p,p'-DDD, p,p'-DDT), and for the PCBs congeners (PCB-52, -101, -138, -150, and -180). In all species of farm animals, the most frequently detected pollutants were p,p'-DDE (80-100% of samples), HCB (73.9-100%), and gamma-HCH (69.6-100%). Species differences in the concentrations of HCB, alpha-, beta-, and gamma-HCH, dieldrin, p,p'-DDE, p,p'-DDD, and PCBs were noted as well as differences in the concentrations of some isomers of HCH, DDT-related chemicals, and PCB congeners in the same species.

Oestrogen and progesterone concentrations in plasma and oviductal tissue of ewes exhibiting a natural or induced oestrus.

Synchronisation of oestrus in Karagouniki ewes by administration of the standard dose of progesterone results in lower fertility than observed when these ewes ovulate naturally. This suggests that the optimum dose of progesterone may be breed dependent. The exogenous progesterone may perturb the concentrations of oestradiol-17beta and progesterone in blood plasma and the oviductal wall. This possibility was investigated using Karagouniki ewes allocated at random to three treatments (n=4 per treatment). Ewes were allowed to exhibit natural oestrus (N) or oestrus was synchronised by administration of 250 mg (LP) or 375 mg (HP) progesterone (subcutaneous implants) followed by PMSG at 8 mg/kg live weight i.m. 14 days later. Oestrus was observed using teaser rams. Blood samples were collected for plasma oestradiol-17beta and progesterone assay from the onset to the end of oestrus at 2 h intervals. The uterus of each ewe was recovered at the end of oestrus and samples of the oviductal wall were taken from both oviducts and prepared, separately, for progesterone and oestradiol-17beta assay. Statistical analysis was performed using univariate analysis of variance. Plasma oestradiol-17beta concentrations from the onset to the end of oestrus were highest for N ewes and lowest for HP ewes with the values for LP ewes occupying an intermediate position. The differences were significant (P<0.05) between HP and the other two treatments from 4 to 12 h after the onset of oestrus and then between all treatments until the end of oestrus. Plasma progesterone levels were similar and fairly constant from the onset to the end of oestrus for N and LP. The plasma progesterone levels for HP were significantly (P<0.05) higher than for the other two treatments throughout oestrus. In oviductal wall samples, the oestradiol-17beta concentration was significantly (P<0.05) higher for N ewes than for synchronised ewes and the levels were similar for LP and HP ewes. The concentration of oestradiol-17beta differed (P<0.05) between right and left oviducts for N ewes but not for ewes of either of the synchronised oestrus treatments. Progesterone concentrations in oviductal wall samples were highest (P<0.05) for HP ewes and the values for N and LP ewes were similar. The concentration of progesterone did not differ between right and left oviductal wall samples within treatments. It was concluded that the higher dose of exogenous progesterone perturbed the levels of oestradiol-17beta and progesterone in blood plasma and the oviductal wall, and this could explain the lower levels of fertility (relative to naturally occurring oestrus) observed when this protocol is used for Karagouniki ewes in practice.

Effects of dietary vitamin A intake on acrosin- and plasminogen-activator activity of ram spermatozoa.

Acrosin and plasminogen activators are proteolytic enzymes of ram spermatozoa that play an essential role in the induction of the acrosome reaction, as well as the binding of spermatozoa to the oocyte and their penetration through the layers that surround the oocyte. Since vitamin A can alter gene expression in various tissues, testis included, this study was undertaken to evaluate the possible effect of vitamin A intake on acrosin- and plasminogen-activator activity. During a 20-week experiment, 15 rams of the Greek breed Karagouniki, divided to three groups, received different amounts of vitamin A per os in retinyl acetate capsules (group A, controls, 12,500 iu/animal per day; group B, 50,000 iu/animal per day; group C, 0 iu/animal per day up to the 13th week, then 150,000 iu/animal per day until the end of the experiment). Acrosin- and plasminogen-activator activity were determined by spectrophotometric methods. Vitamin A was determined in blood plasma by HPLC. No statistical differences were detected regarding the body weight of the rams or the qualitative and quantitative parameters of their ejaculate throughout the whole experiment. No statistically significant alterations of enzyme activity were detected in group B. In group C, both enzyme activities started declining in week 9. Compared with controls, maximum reduction for acrosin was 49% on week 11 and for plasminogen activators 51% in week 14. Activities returned to normal rates after vitamin A re-supplementation. To date, the main result of vitamin A deficiency was known to be arrest of spermatogenesis and testicular degeneration. A new role for vitamin A may be suggested, since it can influence factors related to male reproductive ability before spermatogenesis is affected.

Synchronization of ovulation and fixed time intrauterine insemination in ewes.

A novel method for oestrus-ovulation synchronization in sheep followed by fixed time insemination is presented herewith. Mature dry ewes (n = 28) of Karagouniko breed being at an unknown stage of the oestrous cycle, were used during the middle of breeding season. The treatment protocol consisted of an initial administration of a GnRH analogue followed 5 days later by a prostaglandin F2alpha injection. Thirty-six hours later a second GnRH injection was administered to synchronize ovulation, and laparoscopic intrauterine insemination was performed 12-14 h later. Three days after insemination, fertile rams were introduced into the flock twice daily and oestrus-mating detection was carried out. For progesterone (P(4)) determination, blood samples were collected on alternate days, starting 2 days before the first GnRH injection and continuing for 17 days after insemination. An additional sample was taken on the day of insemination. Pregnancy diagnosis was carried out by trans-abdominal ultrasonography. Fourteen ewes (50%) conceived at insemination and maintained pregnancy; from the remainder 14 ewes 10 became pregnant at natural service, while four, although they mated at least two to three times, failed to conceive. In response to the first GnRH, P(4) concentration increased at higher levels in ewes that conceived at AI compared with those that failed to conceive (47.54 and 22.44%, respectively; p < 0.05). Significant differences (p < 0.05) in mean P(4) concentration between pregnant and non-pregnant animals were detected 1 day before AI (0.17 +/- 0.06 and 0.26 +/- 0.14 ng/ml, respectively) on the day of AI (0.15 +/- 0.04 and 0.24 +/- 0.08 ng/ml, respectively) as well as 9 and 11 days thereafter (0.48 +/- 0.12 and 0.38 +/- 0.12 ng/ml; 0.68 +/- 0.14 and 0.50 +/- 0.18 ng/ml, respectively). These results indicate that using the proposed protocol, an acceptable conception rate can be achieved which could be further improved by modifying the time intervals between interventions.

Covalent binding of an NAD+ analogue to horse liver alcohol dehydrogenase in a ternary complex with pyrazole.

Examination of the model of the fixation site of the adenosine phosphate part of NAD+ on horse liver alcohol dehydrogenase led us to synthesize a NAD+ analogue N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]-NAD+ in order to alkylate the carboxylic acid group of Asp-273 and to convert the normally dissociable coenzyme into a permanently bound prosthetic group. This NAD+ analogue is coupled to the horse liver alcohol dehydrogenase in the ternary complex formed with pyrazole. In these conditions the degree of fixation varies between 0.4 and 0.58 coenzyme molecule/enzyme subunit molecule. The N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]NAD+ acts as a true prosthetic group which can be reduced and reoxidized by a coupled substrate reaction and the internal activity of this holoenzyme corresponds to the amount of analogue incorporated.

Alkylation of 6-phosphogluconate dehydrogenase from Candida utilis with coenzyme analogues.

The mechanism of the inactivation of 6-phosphogluconate dehydrogenase from Candida utilis with two coenzyme analogues can be differentiated on the basis of kinetic studies and of the properties of the inactivated enzyme. 3-Chloroacetylpyridine--adenine dinucleotide phosphate is clearly an affinity label and 3-choloroacetylpyridine--adenine dinucleotide a second-order reagent. For 3-chloroacetylpyridine--adenine dinucleotide phosphate, there is a loss of one thiol per subunit at complete inactivation whereas for 3-chloroacetylpyridine--adenine dinucleotide 2.7 thiol groups are lost. The fluorescence of the protein is quenched after alkylation by 3-chloroacetylpyridine--adenine dinucleotide phosphate and there is no quenching after the inactivation with 3-chloroacetylpyridine--adenine dinucleotide.

Reaction of liver alcohol dehydrogenase with halogenoacids. Fate of the iodide anion released by carboxymethylation and enzymic catalysis of iodide solvolysis.

The fate of the iodide liberated during carboxymethylation of Cys-46 in horse liver alcohol dehydrogenase has been determined with 125I-labeled iodoacetate. The [125I]iodoacetic acid was prepared from mesyloxyacetic acid and sodium [125I]iodide. When carboxymethylation of the enzyme is carried out in solution or in the crystalline state, no iodide is bound to the protein. The rate of iodide during the reaction of iodoacetate, determined with an iodide-specific electrode, has been found to be biphasic: the fast phase corresponds to the carboxymethylation and the slow phase to iodide liberation due to the presence of protein. With 3-iodopropionate (2.5 mM), no inactivation was detected, but in the presence of the enzyme, 10 equivalents of iodide were liberated per subunit in 1 hr. NADH does not inhibit this reaction. The electron density attributed to an iodide bound to the zinc atom of the crystalline enzyme is reinterpreted in view of these results as due to an imidazole bound to the active-site zinc. In the carboxymethylation, the reactivity of bromoacetate is higher than that of iodoacetate.

Degradation of chlorophenols by Phanerochaete chrysosporium: effect of 3,4-dichlorophenol on extracellular peroxidase activities.

Extracellular peroxidases play an important role in the degradation of chlorophenols by Phanerochaete chrysosporium. Depending on the moment of 3,4-dichlorophenol addition, the production of lignin peroxidase and manganese peroxidase in C-limited agitated cultures was affected in opposite ways. In cultures that received 3,4-dichlorophenol at the time of inoculation, fungal growth was reduced and peroxidases were not produced, whereas peroxidase activities were stabilized after 3,4-dichlorophenol addition to pregrown cultures. Further investigation revealed that mRNA encoding lignin peroxidase was not produced in cultures started with 3,4-dichlorophenol, suggesting that the onset of secondary metabolism was affected. In addition, the stabilization of lignin peroxidase activity was not the result of an activation of lignin peroxidase gene transcription, as shown by Northern blot experiments, but likely due to the inhibition of peroxidase degradation by extracellular proteases.

Dependence of the substrate specificity and kinetic mechanism of horse-liver alcohol dehydrogenase on the size of the C-3 pyridinium substituent. 3-Benzoylpyridine-adenine dinucleotide.

The kinetic mechanism and the substrate specificity of liver alcohol dehydrogenase are changed when 3-benzoylpyridine-adenine dinucleotide is used as coenzyme. Only primary alcohols are substrates of the enzyme and with ethanol the mechanism becomes rapid-equilibrium random bi-bi. According to model building experiments on a graphic display, the benzoyl group partially enters the substrate binding site, whereas the essential interactions between coenzyme and enzyme are preserved. This restraint on the substrate binding site provides a molecular explanation for the observed dependence between coenzyme and substrate chemical structures.

The effect of melatonin implants on blood testosterone and acrosin activity in spermatozoa of the ram.

In a series of consecutive blood sampling in 15 days intervals over 15 weeks after implantation of melatonin in rams an increased mean value, basal level and number of peaks of testosterone was observed in samples of the third fortnight (45th day). This increase was greater in the autumn (breeding season) than in spring (non-breeding season). Total acrosin activity in spermatozoa was increased between days 35-56 (autumn) and days 49-70 (spring) after implantation and the relative increase was higher in autumn than in spring. The increase of acrosin activity was independent of the changes of testosterone. An increase of acrosin activity by melatonin, in cases of low activity, might improve fertilization rates in sheep not only during the breeding season, but also during the non-breeding season (after oestrus induction).