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The present study aimed to investigate the impact of age, season and ejaculation on ram testicular blood flow and echotexture. The survey was conducted biweekly on 7 Chios rams for one year, including breeding and non-breeding periods. The rams were divided into 2 age groups: 3 rams 2-6 years old (mature) and 4 rams 9-13 years old (old). Hemodynamic indices [Pulsatility index (PI), Resistive index (RI), End-diastolic velocity (EDV), testicular artery Diameter (D), Time-averaged maximum velocity (TAVM), Blood flow volume (BFV)] and echotexture parameters [Mean value (MV), Contrast (Con), Gray value distribution (GVD), Run length distribution (RunLD), Long run emphasis (LRunEm), Entropy (Ent), Correlation (Cor), Standard deviation (StD), Gray variance (GV) and Gradient mean value (GMV)] were evaluated in each testis before and after ejaculation. Ejaculation did not affect testes blood flow or echotexture (p0.05). PI and RI were higher in the breeding period compared to the non-breeding period, for both testes (p⟨0.001). Left testis GV and Cor before ejaculation were lower (p=0.01) and higher (p=0.03), respectively, in the breeding compared to the non-breeding period. Left testis D (p=0.005) and BFV (p⟨0.001) were higher in old compared to mature rams after ejaculation. Right testis Con (p=0.03) and Cor (p=0.05) before ejaculation were higher in old rams, whereas right testis Ent after ejaculation was higher in mature rams (p=0.05). In conclusion, testicular blood flow and echotexture are affected by season and ram age, but not by ejaculation.

Farm animals behavior research uses video cameras, mainly for visual observation and recording. The purpose of this feasibility study was to enrich the predictable methods of boar semen production capacity by correlating sperm variables with the scrotal contractions (SC) frequency and intensity. A video camera was used to record the reaction of the scrotum during ejaculation. The respective collected ejaculates were evaluated and semen parameters, such as viability, morphology, membranes functional integrity and kinematics, were determined. The camera recorded the scrotal contractions/relaxations and the video was handled by the Image Processing Toolbox of Matlab (Mathworks Inc., Natick, MA, USA). The SC intensity was verified as a percentage change in the scrotum size among the video frames of maximum contraction and relaxation. The archived data from the frames were analyzed statistically, using a linear mixed effects model that involved sperm assessed parameters. Correlations of the SC intensity with the average path velocity, VAP (R2 = 0.591, p = 0.043) and with the percentage of the cytoplasmic droplets (R2 = 0.509, p = 0.036) were noticed. Previous studies reported the positive correlation of VAP with the number of live-born piglets. In conclusion, video monitoring of the boar scrotal function during ejaculation is useful, but more research is needed to establish its appropriateness as a supplementary method for the prognosis of boar ability to produce high-quality semen.

The objective of the study was to investigate the effect of porcine circovirus type 2 (PCV2) vaccination in piglets on sperm quality of young boars. A total of 136 sows were divided in four groups of 34 animals each (17 vaccinated with Circovac®, Merial and 17 unvaccinated in each group). A total of 1 200 piglets were selected, half of which were vaccinated against PCV2 on the 21st day (Porcilis® PCV, MSD) and the other half was left unvaccinated. Four groups of 300 pigs each were formed as follows: PS group (vaccinated sows + piglets), S group (vaccinated sows + unvaccinated piglets), P group (unvaccinated sows + vaccinated piglets), C group (unvaccinated sows + piglets). Furthermore, 80 boars (20 piglets per group) were selected and slaughtered at the age of 5.5 months and weight of 95 ± 5.5 kg and their epididymal sperm was collected and evaluated for motility and kinetics, concentration and morphology. Additionally, 10 pigs from each group were used for blood sampling and serological testing for PCV2 IgM and IgG antibodies at the age of 21, 70, 110 and 150 days. The IgG and IgM patterns suggested that the piglets were coming into contact with PCV2 early in life. The S group demonstrated significantly lower curvilinear velocity (VCL, μm/s), amplitude of lateral head displacement (ALH, μm) and significantly higher head abnormalities (%) compared to other groups (P< 0.05). In conclusion, vaccinated young boars showed some improved epididymal sperm kinetic indices and head morphology.

Simple Summary Artificial insemination with extended liquid boar semen is widely used in the swine industry. The identification of the relationship between boar sperm characteristics and fertility could be of substantial importance to reproduction management. This study evaluated the relationship between boar sperm characteristics and sperm/seminal plasma proteins with main parameters of field fertility. Immotile spermatozoa and spermatozoa with biochemically active plasma membranes affected the number of live-born piglets and litter size of ≥12 piglets. The proteins osteopontin 70 and glutathione peroxidase 5, both separately and in combination, affected the farrowing rate. The combination of immotile sperm and protein osteopontin 70 explained the variation regarding litter size with ≥12 piglets. In conclusion, the evaluation of semen quality variables combined with the evaluation of specific sperm or seminal plasma proteins could provide useful information on in vivo fertilizing capacity of semen doses. Abstract This study aimed to evaluate boar sperm characteristics and proteins, in relation to their importance regarding in vivo fertility. Sixty-five ejaculates were used and 468 sows (parity ≥ 2) were inseminated. Sperm CASA kinetics, morphology, viability, DNA fragmentation, mitochondrial membrane potential, sperm membrane biochemical activity (HOST) and sperm proteins (Heat Shock Protein 90-HSP90, glutathione peroxidase-5-GPX5, Osteopontin 70-OPN70) were assessed and related to field fertility (number of live-born piglets—NLBP, litter size ≥ 12 piglets—LS, farrowing rate—FR). Statistical analysis was conducted with simple and multiple regression models. Simple regression analysis showed that immotile sperm (IM) significantly affected the NLBP and LS, explaining 6.7% and 6.5% of their variation, respectively. The HOST positive spermatozoa significantly affected the NLBP and LS, explaining 24.5% and 7.8% of their variation, respectively. Similarly, sperm with activated mitochondria significantly affected the NLBP, explaining 13.5% of its variation. Moreover, the OPN70 affected LS and FR, explaining 7.5% and 10.8% of their variation, respectively. Sperm GPX5 protein affected FR, explaining 6.7% of its variation. Multiple regression analysis showed that the combination of IM and/OPN70 explains 13.0% of the variation regarding LS, and the combination of GPX5 and OPN70 explains 13.6% of the variation regarding FR. In conclusion, the estimation of parameters IM, membrane biochemical activity, mitochondrial membrane potential, OPN and GPX5 can provide useful information regarding semen doses for field fertility.

The objective of the study was to investigate the efficiency of three enrichment methods to separate boar spermatozoa. Twenty-four ejaculates from 12 boars (2 ejaculates/boar) were extended (30 × 106 spermatozoa/mL) in commercial Beltsville Thawing Solution. Each semen sample was processed with glass wool column (GW) and glass beads (GB) filtration and with the single-layer centrifugation (SLC) technique. Semen samples before (control; C) and after treatment were evaluated for sperm CASA motility/kinetics and concentration, viability, morphology and chromatin integrity. Data were analysed with mixed models. The concentration of total and motile spermatozoa was significantly decreased after treatment in groups GW and SLC, but not in group GB. Group GW showed increased values of WOB compared with both groups C and GB. Group GB showed greater values of rapid movement spermatozoa and lower values of slow movement spermatozoa compared with group C. In group SLC, higher values of VSL, LIN and STR were observed compared with group C. In conclusion, all techniques under examination enhanced various CASA variables. Based on our results, the GB method is a promising alternative separation technique for boar sperm and deserves further research regarding swine in vitro fertilization.

The aim of the study was to investigate the effect of iron oxide (Fe) and silver (Ag) nanoparticles (NPs) on ram semen. A skim milk extender without antibiotics was used as a diluent of 21 ejaculates (8 rams, 2–3 ejaculates/ram). The groups of control (C, semen without NPs), Fe NPs (3.072 mg Fe3O4/mL semen), and Ag NPs (2.048 mg Ag-Fe/mL semen) were incubated (15 °C, 30 min), and then a magnetic field was used for NPs’ removal. Standard microbiological procedures were performed for all groups. Post-treated samples were stored (15 °C) for 24 h, and sperm variables (kinetics by computer assisted sperm analysis (CASA), viability, morphology, HOST, DNA integrity) were evaluated at 6 and 24 h. Semen data were analyzed by a mixed model for repeated measures and microbiological data with Student’s t-test for paired samples. At 6 h of storage, VCL and rapid movement-spermatozoa, and at 24 h, total/progressive motility and amplitude of lateral head displacement (ALH) were significantly decreased in group Ag compared to control. In group Fe, progressive/rapid movement-spermatozoa were significantly lower compared to control after 24 h of storage. Only in group Ag was a significant reduction of total bacterial count revealed. In conclusion, the examined Fe NPs demonstrated slight antibacterial effect, while the examined Ag NPs provided higher antibacterial properties accompanied by cytotoxicity.

The aim of this study was to investigate the effect of experimental Toxoplasma gondii infection on ram sperm quality. Five months old, pre-pubertal, rams were divided into four groups (n = 8 per group). Group A was the control group, the remaining animals received per os (p.o.) 5000 oocysts per ram. Group B did not receive treatment post-infection (p.i.). Group C received sulphadimidine (intermuscular injection (i.m.) 33 mg/kg for eight days, every 48 hrs) two months p.i. and Group D received the same drug twice (24 hours p.i. and two months later). Blood samples were collected every 15 days to detect serum immunoglobulin G (IgG). Epididymal sperm samples were analyzed for concentration, kinetics, morphology/viability, functional membrane integrity, DNA integrity, and the presence of parasite DNA. Histopathological examination was performed on the testes. The IgG titres in infected groups raised two weeks p.i. and remained high for four months. Higher values were noticed in viability and functional membrane integrity in positive spermatozoa in the control group compared to other groups, level of significance p &lt, 0.05. Abnormal sperm was higher in groups C and D vs. A and C vs. B (p &lt, 0.05). T. gondii DNA was detected in three sperm samples of the infected rams (12.5%). Histopathology revealed similar findings with little variation among all infected groups, characterized mostly by increased interstitial connective tissue, non-purulent inflammation, and presence of seminiferous tubules with spermatogenic cell depletion, which increased gradually from D to C and B groups. In conclusion Toxoplasmosis in pre-pubertal age negatively affected mature ram sperm quality, while sulphadimidine administration failed to alter this.

This study examined the effect of Fe3O4 nanoparticles on boar semen. Beltsville thawing solution without antibiotics was used to extend ejaculates from 5 boars (4 ejaculates/boar). Semen samples of control group (C) and group with Fe3O4 (Fe, 0.192 mg/mL semen) were incubated under routine boar semen storage temperature (17 &deg, C) for 0.5 h and nanoparticles were removed by a magnetic field. Before and after treatment, aliquots of all groups were cultured using standard microbiological methods. The samples after treatment were stored (17 &deg, C) for 48 h and sperm parameters (computer-assisted sperm analyzer (CASA) variables, morphology, viability, hypo-osmotic swelling test (HOST), DNA integrity) were evaluated at storage times 0, 24, 48 h. Semen data were analyzed by a repeated measures mixed model and microbial data with Student&rsquo, s t-test for paired samples. Regarding CASA parameters, Fe group did not differ from C at any time point. In group C, total motility after 24 h and progressive motility after 48 h of storage decreased significantly compared to 0 h. In group Fe, linearity (LIN) after 48 h and head abnormalities after 24 h of storage increased significantly compared to 0 h. The microbiological results revealed a significant reduction of the bacterial load in group Fe compared to control at both 24 and 48 h. In conclusion, the use of Fe3O4 nanoparticles during semen processing provided a slight anti-microbiological effect with no adverse effects on sperm characteristics.

Mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) can negatively affect pig health. However, little is known about their effects on boar semen. We assessed the individual and combined effects of DON and ZEN on boar semen in vitro. In a pretrial, we determined the minimum dose (MiD) of each mycotoxin that induces a significant alteration of sperm progressive motility, as investigated using computer-assisted semen analysis (CASA). In the main trial, the individual and combined effects of each mycotoxin&rsquo, s MiD on sperm motility and kinetics (CASA analysis), morphology (SpermBlue staining), viability (calcein-propidium iodide staining), membrane functional status (hypoosmotic swelling test), and chromatin integrity (acridine orange staining) were analyzed. Pretrial results suggested a MiD of 50.6 &mu, M and 62.8 &mu, M for DON and ZEN, respectively. In the main trial, DON and ZEN administered at MiD significantly affected CASA parameters (e.g., increase of immotile spermatozoa, reduction of progressive motile spermatozoa), decreased sperm viability, and affected sperm morphology (head abnormalities) and membrane functional status. DON and ZEN showed less than additive effects on most parameters tested and a synergistic effect on viability and on two CASA parameters. In conclusion, DON and ZEN showed individual and combined toxic effects on boar semen in vitro.

ΔΕΝ ΔΙΑΤΙΘΕΤΑΙ ΠΕΡΙΛΗΨΗ, The intensification of sheep production, by permanent genetic selection and the development of breeding technology, has led to the creation of highly productive sheep breeds. In this way, many highly productive breeds were created which could demonstrate their high production potentials only under perfect conditions of nutrition, accommodation and care. Preservation of indigenous breeds is of great importance in order to protect and safeguard those breeds and, in this way, it is possible to restore some of the characteristics that are lost during intensive selection, which are mostly related to resistance. The Racka sheep (Serbian: Vitoroga žuja) is considered to be an autochthonous breed and a genetic resource in the Republic of Serbia. As a primitive breed with low productivity, it offers no economic profitability and, thus, there is no great interest in its breeding. According to the FAO data from 2008-2014, the number of these sheep ranges from 500 to 1000. The objective of this study was to determine the phenotypic variability and to assess the external measurements of the Racka sheep. One-hundred fifty Racka breed ewes were included in this study. The effects of three farms on the phenotypic characteristics and their body indexes were calculated. The significance of the research is reflected in the advancement of this breed and in the assessment of the possibilities of selection work in these herds.

The aim of the study was to investigate the effect of the antioxidant astaxanthin on boar semen. Twenty ejaculates from 10 boars (two ejaculates/boar) were extended and split in three groups: semen control (SC), solvent control (C; semen with dimethyl sulfoxide, the diluent of astaxanthin) and semen with astaxanthin (A) in concentration 0.5 μmol/L. Sperm quality parameters (motility and kinetics, morphology, viability, functional integrity of sperm plasma membrane by Hypo-Osmotic Swelling Test [HOST] and DNA integrity) were assessed at 0, 24 and 48 hr of storage at 17°C (experiment I), before (0 hr) and after (1 hr) of sperm thermal resistance assay at 37°C (experiment II) and finally before (0 hr) and after (1 hr) sperm in vitro incubation (38.5°C, 5% CO2 , maximum humidity [experiment III]). In experiment I, group A performed overall better than group SC and as a tendency better than group C regarding viability. Total motility, rapid spermatozoa and HOST remained constant across time in group A, whereas they decreased in the remaining groups. In experiment II, regarding motility and viability, group A displayed better results across time than the other two groups. In experiment III, viability and total motility decreased in groups SC and C, while in group A, these parameters were not significantly different between the examination time points. In conclusion, astaxanthin has a beneficial and protective effect on boar semen quality under the investigated conditions.

The present study aimed to provide new and objective information regarding the effect of season and age on the characteristics of Chios ram semen. The semen collection was performed biweekly throughout a year, including both breeding and non-breeding periods. Seven active Chios rams were divided in two age groups: three rams of 2–6 years old (mature) and four rams of 9–13 years old (old). After collection, semen volume and sperm concentration were measured. One hundred sixty-eight (168) semen samples were collected, extended and evaluated. All samples were tested for motility and kinetics (Computer Assisted Sperm Analyzer), viability-membrane integrity (Eosin-Nigrosin), morphology (SpermBlue®), chromatin integrity (Acridine Orange Test) and sperm membrane biochemical functionality (Hypo-osmotic swelling test, HOST). Motility did not differ significantly between periods (p > 0.05) or between age groups (p > 0.05). The percentage of hyperactivated spermatozoa and head abnormalities was higher in the non-breeding compared to the breeding period (p = 0.011 and p = 0.001, respectively). Tail abnormalities were higher in the breeding (p = 0.022) and HOST + spermatozoa were lower in the non-breeding period (p

The aim of this study was to evaluate the effect of antioxidant agents and freezing methods on the ability of ram sperm to preserve its post-thaw quality characteristics. Six Chios rams were subjected to 52 weekly semen collections. Each ram was used as semen donor for freezing experiments once every 2 weeks. Equal number of good quality spermatozoa from each ejaculate (concentration ≥1 × 109 spermatozoa/ml, motility ≥70%, motility score ≥3.5) were pooled. Three equal aliquots of the pooled sample were diluted using three different fractions of a milk-based and glycerol extender (control, quercetin-enriched, α-tocopherol-enriched). Three freezing methods were applied (slow and fast freezing rate in a programmable freezer, vapors of liquid nitrogen) in every aliquot. Sperm aliquots were tested before freezing, immediately after thawing and after 3 h of incubation at 37 °C. Sperm motility (%) was evaluated microscopically. The percentage of membrane and acrosome-intact spermatozoa (IL%) as well as the percentage of membrane-intact and acrosome-reacted spermatozoa (ARL%) were determined by eosin-nigrosin stain. Furthermore, the percentage of hypo-osmotic swelling (HOS) test-positive spermatozoa was estimated. The results revealed no beneficial effect of the antioxidant treatment on the parameters of post-thaw semen (P > 0.05). However, the slow freezing rate method was more beneficial regarding motility, IL, ARL and HOS-positive spermatozoa compared to the other methods. In conclusion, the antioxidant agents used in this study failed to protect sperm against cryopreservation stress; however, the choice of the appropriate freezing method could contribute to the improvement of post-thaw ram sperm quality.

The computer-assisted sperm analysis (CASA) has become a standard laboratory tool. Although it contributes a lot to the objective sperm motility assessment, its measurements may be affected by many factors. The aim of the study was to evaluate the effect of chamber on boar semen CASA results. Totally, 100 extended (30 × 106 sperm/ml) boar semen samples were analysed by CASA. Each sample was evaluated using Makler, Leja 4 chamber 20 μm and conventional glass slide/coverslip chambers (MC, LC and GSC, respectively). The differences in values between MC and LC and between MC and GSC were significantly positive (higher values for MC compared with LC and GSC) for total motility, progressive, rapid movement, VCL, VSL, VAP, STR and hyperactive, thus indicating a systematic effect. Between LC and GSC, the differences in many parameters (non-progressive, progressive, slow, LIN, STR, hyperactive) were evenly distributed around zero, while in all other parameters the differences were significantly positive (higher values for LC compared with GSC), except for medium movement. Based on the estimated intraclass correlation coefficients, the method agreement between MC and LC and between LC and GSC was overall moderate to good, depending on the parameter; nonetheless, it was poor between MC and GSC. The limits of agreement between methods can vary considerably depending on the parameter and should be considered when comparisons between CASA measurements of different andrology laboratories or studies have to be performed.

BACKGROUND Zearalenone (ZEN) is a mycoestrogen with a ubiquitous presence in animal feeds, which also has hematotoxic, hepatotoxic, nephrotoxic, and immunotoxic properties. However, there is a paucity of literature that discusses the effects of ZEN on rabbits. OBJECTIVES The aim of this study was to evaluate the effect of a prolonged, low-level (50 μg ZEN/kg body weight) exposure on the clinicopathologic and redox status analytes of rabbit bucks. METHODS Ten adult bucks were included in the study. Each underwent a 7-week control period, followed by a 7-week exposure period. Water or ZEN solutions were daily administered orally (0.5 mL) during the control and exposure periods, respectively. Blood samples were collected weekly for Complete Blood Counts, serum biochemical analyte and reactive oxygen metabolite (ROM) measurements. Data were analyzed using a mixed model, and the level of significance was set at a P of

The computer-assisted sperm analysis (CASA) has become a standard laboratory tool. Although it contributes a lot to the objective sperm motility assessment, its measurements may be affected by many factors. The aim of the study was to evaluate the effect of chamber on boar semen CASA results. Totally, 100 extended (30 × 10

Porcine intra cytoplasmic sperm injection’s (ICSI) efficacy by selected protocol steps was investigated. Three trials per year’s period (hot, medium, cold) were carried out. Only large size follicles (6-8mm) were aspirated, brilliant cresyl blue (BCB) test was performed and only the BCB+ oocytes were in vitro maturated (40h) and involved to ICSI process. The presumptive embryos were in vitro cultured (15h). Raw boar semen and SpermCatch® as slowing medium were used. No differences were observed between periods regarding early embryonic development and maturation competence. ICSI achieves acceptable porcine early embryonic development rates under the investigated conditions.

The aim of the study was to determine whether the presence of astaxanthin (ASX) protects boar spermatozoa against damage related to cryopreservation. Pooled ejaculates extended in Beltsville Thawing Solution (BTS) were used. Three experiments were conducted: 1) sperm samples were pre-incubated overnight (17 °C) with ASX (0, 0.5, 5, 15 μM) prior to freezing and then frozen using cooling and thawing extenders supplemented with ASX (0, 0.5, 5, 15 μM); 2) sperm samples were treated with ASX (0, 0.5, 5, 15 μM) only during overnight pre-incubation (17 °C) prior to cryopreservation; and 3) a thawing extender was supplemented with ASX (0, 0.5, 5, 15 μM). The groups were as follows: control (C; no treatment), ASX 1 (0.5 μM), ASX 2 (5 μM) and ASX 3 (15 μM). Total (TM) and progressive (PM) motility was analyzed using CASA, while sperm viability, reactive oxygen species generation, lipid peroxidation and apoptoticlike changes were analyzed using flow cytometry. Sperm variables were evaluated prior to freezing as well as 30 and 150 min after thawing. In Experiment 1, the values of TM and sperm viability post-thaw were less in the ASX 3 than C group. In Experiment 2, there was no effect of ASX on any of the sperm variables evaluated, while in Experiment 3, apoptotic-like changes were less in the ASX 1 than C group. In conclusion, there was a subtle beneficial effect on cryopreserved boar spermatozoa after addition of ASX to thawing media.

Swine seasonal infertility reduces the productivity and profitability of a pig farm. The main causes of this condition are elevated environmental temperatures and long photoperiod during the summer season. The aim of this study was to investigate which sperm proteins and parameters are affected during the period of seasonal infertility. Depending on the environmental temperatures, the period from October to June was considered as cold and the period from July to September as warm season. A total of 65 ejaculates from 18 boars were collected over a year. Each semen sample was evaluated for kinetics (Computer Assisted Semen Analyzer), morphology (Sperm Blue stain), viability (Propidium Iodide - Calcein AM stain), mitochondrial membrane potential (Rhodamine 123 – Propidium Iodide stain), membrane integrity and functionality (Hypo-osmotic swelling test) and sperm DNA integrity (Acridine Orange Test). Moreover, selected proteins (HSP90, GPX5, OPN) were detected and quantified. The kinetic parameters VSL, LIN and the midpiece abnormalities were significantly higher in the warm compared to the cold season (p

OBJECTIVE To determine the effect of subchronic oral exposure to zearalenone (ZEA) at a daily dose of 50 μg of ZEA/kg of body weight (an environmentally relevant concentration) on the reproductive system of rabbit bucks. ANIMALS 8 healthy sexually mature New Zealand White rabbits. PROCEDURES During the experimental period (March to June), each rabbit underwent a 7-week control protocol and then a 7-week treatment protocol. Water (0.5 mL) or ZEA solution (50 μg/kg [0.5 mL]) was administered orally once daily during the control and treatment period, respectively; ejaculates were collected weekly. Studied end points included semen quality variables (spermatozoa kinetics, morphology, viability, and DNA fragmentation), serum testosterone concentration, and results of histologic examination of the testes and epididymides following euthanasia at the end of the experimental period. RESULTS Treatment with ZEA solution resulted in significant increases in spermatozoa beat-cross frequency, in the percentages of spermatozoa with head and midpiece abnormalities, and in the percentages of DNA-fragmented spermatozoa, compared with effects of the control treatment. Serum testosterone concentration, other spermatozoa velocity variables, and percentages of progressive and total motility, rapidly or slowly moving spermatozoa, and live spermatozoa did not differ significantly between the 2 periods. Histologic examination revealed no patterns of abnormal findings in the testes and epididymides. CONCLUSIONS AND CLINICAL RELEVANCE Oral treatment with ZEA solution at an enviromentally relevant concentration caused minor interference with rabbit bucks' sperm quality. Although mostly considered mild, the sperm quality changes warrant further investigation in terms of fertilizing capacity impairment.

This study investigated correlations among ram semen quality parameters, testicular vascularity and echotexture. Measurements were performed biweekly for a year, before (BE) and after ejaculation (AE), on seven Chios breed rams [three rams (group A) and four rams (group B) of 2-6 and 9-13 years old, respectively], considering breeding (1) and non-breeding period (2). Hemodynamics [Pulsatility Index (PI), Resistive Index (RI)] were evaluated in each testis. Images of testicular parenchyma were analysed to evaluate echogenicity [Mean Value (MV), Contrast (Con), Gray Value Distribution (GVD), Run Length Distribution (RunLD), Long Run Emphasis (LRunEm)]. Semen volume (V), concentration (C), kinetics (CASA), morphology (Sperm Blue), viability (Eosin-Nigrosin) and DNA fragmentation (Acridine Orange) were assessed. In group A/2, semen V was correlated to left testis PI and right testis RI, BE (r = 0.4, p = 0.013/r = 0.6, p

Background Mycoplasma hyopneumoniae (M. hyo) and Porcine Circovirus Type 2 (PCV2) are major pathogens that cause significant health problems in swine worldwide. Maternal derived immunity (MDI) has been suggested as a significant immediate defence factor for newborn piglets and may interfere with piglet’s vaccination-induced immunity. The study aimed to assess the efficacy of a novel combination vaccine (consisting of PCV2 subunits and inactivated M. hyo strain J), against PCV2 and M. hyo natural infection [Porcilis® PCV M Hyo (MSD Animal Health, Boxmeer, the Netherlands)], in the presence of strong maternally derived PCV2 immunity (antibody titre averaged 11.08 log2), under field conditions. The study was performed according to a controlled, randomized and blinded design in a Greek swine unit with Enzootic Pneumonia (EP) and subclinical PCV2 infection. In total, 600 healthy three-week-old suckling piglets were allocated randomly, either to treatment (vaccinated with the test product) or control group (injected with sterile buffered saline). Results Vaccination significantly reduced the severity of lung lesions at slaughter (lesions of cranio-ventral pulmonary consolidation) (P

The objectives of the present study were twofold: (1) to assess the association of body condition score (BCS), β-hydroxybutyric acid (BHBA) and non-esterified fatty acids (NEFA) concentrations in blood serum prior to lambing with the health status of intensively managed dairy ewes during the periparturient period (defined here as 15 days before the expected parturition up to 30 days after lambing), and (2) to evaluate BHBA and NEFA fluctuations and possible association during the latter period. A total of 262 clinically healthy dairy ewes of the Chios breed were used. They were ewe-lambs, primiparous and multiparous animals. Periparturient diseases (pregnancy toxemia, retained fetal membranes, metritis and clinical mastitis) and culling cases (defined as “health disorders”) were recorded. BCS was assessed at 30 ± 3 days before parturition (−30 d). Blood samples were obtained at 30 ± 3 (−30 d) and 15 ± 3 (−15 d) days before the expected parturition as well as 15 (+15 d) and 30 (+30 d) days after parturition; serum BHBA and NEFA concentrations were determined. A multivariable logistic model was developed to assess the effect of BCS, BHBA and NEFA concentrations at 30 d before parturition on “health disorders”. The odds of developing periparturient “health disorders” in normal ewes (BCS 2.75–3.5) at 30 days prepartum were 0.338 times lower when compared with the lean ewes (BCS P = 0.003). In addition, the odds for a unit increase in BHBA is 2.163 to develop periparturient “health disorders” ( P = 0.013). A Linear Mixed Effects model indicated a statistically significant effect of time periods on both BHBA ( P P r = 0.413, P

Contents The objective of this study was to investigate the quality of frozen-thawed semen from different bull breeds. Commercial frozen-thawed bull semen samples (26 per breed, 130 totally) of five breeds (Holstein [Η], Brown Swiss [BS], Limousin [L], Belgian Blue [BB], Blonde d' Aquitaine [BA]) were used. After thawing, each semen sample was subjected to thermal resistance test (TR) for 0.5 and 1 hr at 38°C and hypo-osmotic swelling test (HOST) for 1 hr at 150 mOsm at 37°C. Additionally, all samples were evaluated at times 0 hr (thawing), 0.5 hr (TR), 1 hr (TR) for kinetics by CASA [progressive, immotile, rapid, medium, slow moving spermatozoa, curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), linearity (LIN), straightness (STR), beat cross-frequency (BCF), amplitude of lateral head displacement (ALH), wobble (WOB)]. Moreover, directly after thawing, all semen samples were evaluated for morphometry, morphology, viability and DNA fragmentation. Statistical analysis was conducted using a mixed model for repeated measures. The results showed (a) higher VCL after thawing in H, L breeds compared to BB and BA, (b) higher VAP after thawing in L compared to BB, BA, (c) higher values of progressive spermatozoa after TR in H, BS compared to BB, BA, (d) higher values of rapid spermatozoa after thawing and 0.5 hr of TR in H, BS, L compared to BB, BA, (e) lower viability in BA after thawing compared to H, BS, BB, (f) lower morphological abnormalities in H compared to L, BB, (g) higher head length in Η compared to BB. No significant differences were observed in the results from HOST and DNA fragmentation between breeds. In conclusion, quality characteristics of frozen-thawed bull semen are dependent on the breed. Frozen semen from BB and BA breeds should be handled more carefully after thawing, as it is more sensitive to stress.

Summary Oxidative stress is one of the major factors that contribute to poor semen quality and low rates of in vitro fertilization. Crocetin, a main constituent of saffron (Crocus sativus L.) possesses potent antioxidant activity, by scavenging reactive oxygen species (ROS) and/or enhancing the activity of intracellular antioxidant enzymes. The aim of this study was to investigate, for the first time, the effect of crocetin on the quality characteristics of bull spermatozoa and fertilization rate. For this reason, frozen/thawed bovine spermatozoa were incubated with crocetin (1, 2.5, and 5 μm), for 120 or 240 min, in the presence of a negative control, and evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. In order to evaluate the impact of crocetin on cleavage and blastocyst rate, the compound was added in the IVF medium at the previously identified optimal concentration (2.5 μm). The results indicate that incubation of spermatozoa with 2.5 μm of crocetin resulted in a statistically significant lower production of superoxide anion and hydrogen peroxide, lower lipid peroxidation, and in better maintenance of motility parameters, viability, and acrosomal integrity, with a very small number of cells with DNA fragmentation, compared to the other groups (p

Although the application of modern technologies and techniques in ram semen evaluation is not as widespread as in domesticated animal species, developments have been reported. Improvements in extenders and cryo-protectants have contributed to extended ram semen preservation and better results of ovine artificial insemination. For these reasons, semen evaluation process offering high accuracy and reliability of the results is needed more than in the past. Major advancements have been made on ram semen analysis by using computer assisted semen analyzers. Moreover, evaluation of sperm function in depth as the estimation of sperm mitochondrial status, sperm nucleus chromatin integrity and sperm ability to bind and/or fertilize an oocyte have recently been included to ram semen assessment. This review emphasizes to recent development in these technologies that start to show satisfactory diagnostic value for ram semen evaluation, as well as to their relation with field fertility.

Η κρυοσυντήρηση του σπέρματος του κριού αποσκοπeί στη γeνeτική βeλτίωση μέσω της τeχνητής σπeρματέγχυσης, στην άρση των γeωγραφικών πeριορισμών, στην προστασία απeιλούμeνων μe eξαφάνιση φυλών και στη διατήρηση της βιοποικιλότητας. Η διαδικασία κατάψυξης όμως, eπιφέρeι δομικές, βιοχημικές και λeιτουργικές μeταβολές στα σπeρματοζωάρια. Συγκeκριμένα, βλάπτονται οι κυτταρικές μeμβράνeς και αυξάνeται η διαπeρατότητά τους, μeταβάλλeται η δομή της χρωματίνης τους και προκαλeίται υπeροξeίδωση καθώς παράγονται δραστικές μορφές οξυγόνου. Οι αλλαγές αυτές, eπηρeάζουν αρνητικά τη γονιμοποιητική ικανότητα των σπeρματοζωαρίων του κριού και την πρώιμη eμβρυική ανάπτυξη. Στη βιβλιογραφία eντοπίζονται πολυάριθμeς eπιστημονικές προσπάθeιeς βeλτίωσης της γονιμότητας του κρυοσυντηρημένου σπέρματος του κριού. Πρωταρχικό και κοινό σημeίο διeρeύνησης, υπήρξe η σύνθeση των αραιωτικών μέσων. Τα αραιωτικά του σπέρματος πρέπeι να έχουν, μeταξύ άλλων, κατάλληλη ρυθμιστική ικανότητα και pΗ ώστe να προστατeύουν τα σπeρματοζωάρια από την οσμωτική και τη θeρμική καταπόνηση. Υπό αυτό το πρίσμα, έγινe προσθήκη και μeλέτη πληθώρας ουσιών μe ποικίλeς ιδιότητeς και αποτeλέσματα. Μeταξύ αυτών, χρησιμοποιήθηκαν κρυοπροστατeυτικά που διαπeρνούν (γλυκeρίνη, διμeθυλο-σουλφοξeίδιο) ή όχι (κρόκος αυγού, αποβουτυρωμένο γάλα) την κυτταρική μeμβράνη, σάκχαρα (γλυκόζη, λακτόζη, τρeχαλόζη, ραφινόζη), άλατα (κιτρικό νάτριο, κιτρικό οξύ) και αντιοξeιδωτικά (αμινοξέα, βιταμίνeς, ένζυμα). Άλλοι παράγοντeς που μeλeτώνται eκτeνώς, αφορούν στη θeρμοκρασία και στο χρόνο συντήρησης στα eπιμέρους στάδια της κατάψυξης, στο ρυθμό πτώσης της θeρμοκρασίας και στο πρωτόκολλο αναθέρμανσης. Τα αποτeλέσματα των eρeυνών eίναι eνθαρρυντικά, όχι μόνο ως προς τις διαδικασίeς κατάψυξης/αναθέρμανσης του σπέρματος, αλλά και ως προς τις ωφέλιμeς ιδιότητeς των προσθeτικών. Παρά την πρόοδο που eπιτeύχθηκe μέχρι σήμeρα, eξακολουθούν να υφίστανται κeνά, μέχρις ότου eπιτeυχθeί η διeυρυμένη χρήση του κρυοσυντηρημένου σπέρματος του κριού στην αναπαραγωγική διαχeίριση των ποιμνίων.

Η τeχνητή σπeρματέγχυση (ΤΣ) αποτέλeσe μια από τις σημαντικές κτηνιατρικές eπeμβάσeις για την αύξηση της παραγωγικότητας της παγκόσμιας χοιροτροφίας. Η συνeχιζόμeνη προσπάθeια για τη βeλτίωση των αποδόσeων και τη μέγιστη αξιοποίηση του ζωικού γeνeτικού δυναμικού οδήγησe στην ανάπτυξη και νέων τeχνικών ΤΣ. Μeταξύ αυτών συγκαταλέγονται, η μeτα-τραχηλική ή eνδομητριαία σπeρματέγχυση, η βαθιά μητριαία ή eνδοκeράτια σπeρματέγχυση, η χeιρουργική, λαπαροσκοπική ή μeτά από λαπαροτομή σπeρματέγχυση στους ωαγωγούς, καθώς και διάφορeς άλλeς βιοτeχνολογικές eφαρμογές. Η αποτeλeσματικότητα αυτών των τeχνικών και η πιθανότητα eυρύτeρης eφαρμογής τους αξιολογήθηκαν από πολλούς eρeυνητές μeτά από in vivo πeιραματισμούς. Βέβαια, μέχρι σήμeρα, η αποδοτικότητα, η eύκολη eφαρμογή και το χαμηλό κόστος καθιστούν την παραδοσιακή ΤΣ αναντικατάστατη μέθοδο για τη γονιμοποίηση των συών σe eπίπeδο eκτροφής. Παρ' όλα αυτά, οι νeότeρeς τeχνικές, καθώς και οι διάφορeς βιοτeχνολογικές eφαρμογές αποτeλούν αναγκαία eξέλιξη μe σκοπό τη μέγιστη ωφeλιμότητα από την eκμeτάλλeυση των ζώων υψηλής γeνeτικής αξίας. Από σχeτικούς πeιραματισμούς αποδeικνύeται ότι η eναπόθeση στο κατάλληλο σημeίο της γeνeτικής οδού του θηλυκού και σe συγκeκριμένο χρόνο, eξαιρeτικά μικρού αριθμού σπeρματοζωαρίων, μπορeί να οδηγήσeι σe ικανοποιητικά ποσοστά σύλληψης, τοκeτών και μeγeθών τοκeτοομάδων. Μe μeτατραχηλική σπeρματέγχυση και χρησιμοποίηση του eνός τρίτου της συνήθους δόσης σπέρματος (lxlO 9 σπeρματοζωάρια) eπeτeύχθησαν συγκρίσιμα αποτeλέσματα μe eκeίνα της παραδοσιακής ΤΣ. Αναλογικά, στην eνδοκeράτια σπeρματέγχυση ο αριθμός των σπeρματοζωαρίων που αποδeικνύeται eπαρκής eίναι ακόμη μικρότeρος (150χ10 6 σπeρματοζωάρια), eνώ για τη σπeρματέγχυση στον ωαγωγό eπαρκούν eλάχιστα σπeρματοζωάρια (3χ10 5 ). Η συνeχής βeλτίωση των μέσων που χρησιμοποιούνται στις παραπάνω μeθόδους καθιστά ή τeίνeι να καταστήσeι eφικτή την eφαρμογή τους ακόμη και από μη eξeιδικeυμένο προσωπικό, δίνοντας τη δυνατότητα eπέκτασης των eφαρμογών τους σe στοχeυμένους πληθυσμούς χοιροτροφικών eκμeταλλeύσeων συγκeκριμένων προδιαγραφών. Η ανάπτυξη των νέων τeχνικών ΤΣ δίνeι τη δυνατότητα αξιοποίησης βιοτeχνολογικών προϊόντων, όπως το κρυο-συντηρημeνο και φυλοπροσδιορισμeνο σπέρμα του κάπρου, και της παραγωγής διαγονιδιακών ζώων. Στους στόχους της βιοτeχνολογίας της αναπαραγωγής eντάσσονται και η eνίσχυση της βιομηχανικού τύπου eκτροφής των χοίρων μέσω της βeλτίωσης των αναπαραγωγικών δeικτών, της καλύτeρης αξιοποίησης της τροφής, της βeλτίωσης της σωματικής ανάπτυξης, της ποιότητας του σφαγίου, καθώς και της βeλτίωσης της ανθeκτικότητας στις ασθένeιeς. Τα νeότeρα δeδομένα και τα συμπeράσματα από την eξέλιξη της ΤΣ του χοίρου παρατίθeνται σe αυτήν τη βιβλιογραφική ανασκόπηση-μeλέτη

This study aimed at investigating the in vitro effects of zearalenone (zen) and α-zearalenol (α-zen) on motility and nuclear chromatin integrity (NCI) of boar spermatozoa. Mycotoxins were tested, at levels ranging from 10 to 30 µg ml−1 of diluted semen. Four boars were used for semen collection (eight replicates per boar, four per mycotoxin). After the addition of zen or α-zen, semen samples were incubated for 4 h at 38.5 °C, 5% CO2 and 96% humidified air. Motility and NCI were assessed at 0 and 4 h of incubation. No significant differences were noticed in motility among the experimental groups (P > 0.05) for all tested boars. Chromatin instability was significantly higher (P < 0.05) in spermatozoa of only one boar treated with zen and α-zen independently of the dose. In conclusion, under our experimental conditions, zen and α-zen did not affect the motility of boar sperm, whereas the effects of these toxins on sperm NCI were individual-dependent. Copyright © 2007 John Wiley & Sons, Ltd.

Background The primary objective of this study was to assess the efficacy, under field conditions, of a novel ready-to use Mycoplasma hyopneumoniae (M hyo) and Porcine circovirus type 2 (PCV2) combination vaccine given to piglets as one vaccination (1-shot) at 3 weeks of age. The study was carried out according to a controlled, randomised, and blinded design in a Greek pig herd with clinical M. hyo and subclinical PCV2 infection. Moreover, based on serology at the time of vaccination, the average PCV2 titre was 9.15 log2 and represented the level of maternally derived antibodies (MDA). In total 602 healthy suckling piglets, originating from 4 weekly farrowing batches were allocated randomly, within litters, to one of two groups. The pigs in one group were vaccinated with the test product and the other pigs were injected with saline. Results Vaccination significantly reduced lesions of craneo-ventral pulmonary consolidation in vaccinated group [expressed as lung lesion score (LLS)] (Mixed model ANOVA: p

Σκοπός της παρούσας ανασκόπησης eίναι η παρουσίαση της λeιτουργίας του αυτόματου αναλυτή σπέρματος υποβοηθούμeνου από ηλeκτρονικό υπολογιστή (CASA) και των eφαρμογών του στον τομέα της αναπαραγωγής των παραγωγικών ζώων, ώστe να γίνeι eυρέως αντιληπτή η συμβολή του ως eργαλeίο πρόγνωσης της γονιμότητας των αρσeνικών ζώων Παρατίθeνται οι προϋποθέσeις ορθής λeιτουργίας του αναλυτή, οι παράγοντeς που eπηρeάζουν αυτήν και η αξιολόγηση των eκτιμούμeνων παραμέτρων, μe ιδιαίτeρη έμφαση στην κινητικότητα των σπeρματοζωαρίων. Ο αναλυτής CASA αποτeλeί σημαντικό eφόδιο για τους ασχολούμeνους μe τη διαχeίριση υγeίας των παραγωγικών ζώων, ωστόσο απαιτeίται τήρηση των κανόνων για ορθή eκτίμηση των δeιγμάτων και για αξιοπιστία και δυνατότητα σύγκρισης των αποτeλeσμάτων.

Summary This study aimed to investigate the selective ability of swine zona pellucida (ZP) to bind spermatozoa with normal nuclear chromatin. Ten ejaculates of four boars were used, while hemizona assay was applied for evaluation of binding capability. The results of this study showed that swine ZP has the ability to select spermatozoa with normal chromatin structure for sperm-zona binding process.

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen-thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1-2 years (young) or 4-5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of >or= 2.5 x 10(9) sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell medium and frozen in 0.25 ml straws. The end points of post-thawing semen evaluation were computer-assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per-cell analysis of lipid peroxidation using C11-BODIPY(581/591), sperm-hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen-synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non-capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = -0.63 to -0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.

The effect of porcine or ovine FSH on the maturation rate of porcine oocytes and on the time course of meiotic progression was studied. Groups of 20 grade-A cumulus oocyte complexes, aspirated from slaughterhouse cycling-gilt ovaries, were cultured in vitro in 400 mul of Modified Parker's Medium supplemented with oestrous cow serum and porcine FSH (Folltropin(R)-V, 0.50 mg/ml) or ovine FSH (Ovagen(TM), 0.44 iu/ml), in four-well dishes under mineral oil, at 38.5 degrees C, 5% CO(2) in humidified air. At the end of each 3-h interval, from 3 to 42 h of culture, the nuclear status of oocytes was assessed microscopically (1000x), after fixation (methanol/acetic acid: 3/1) and orcein (2%) staining. Oocytes were classified as (i) immature (IMM), i.e. oocytes at germinal vesicle stage, germinal vesicle break down and prophase I, (ii) metaphase I (MI) and (iii) metaphase II (MII), i.e. oocytes at anaphase I, telophase I and metaphase II. Data were analysed using regression analysis, chi-square and t-test. Nuclear status was assessed in 1610 oocytes (porcine FSH: 787, ovine FSH: 823). Most of the oocytes were at MI from 24 to 33 h (porcine FSH 60.27%, ovine FSH 42.80%, p < 0.001) and at MII from 36 to 42 h (porcine FSH 80.38%, ovine FSH 67.45%, p < 0.01) of culture. Significantly higher maturation rate was observed in porcine FSH than in ovine FSH treated oocytes (86.69 +/- 12.97%, 71.34 +/- 9.86%, mean +/- SD, p < 0.05), after 42 h of culture. In conclusion, under the specific culture conditions, porcine FSH seems to support pig oocyte maturation better than ovine FSH.

This study was conducted to determine the in vitro effects of three different concentrations (125, 187.5 and 250 microM in diluted semen) of zearalenone (zen) and alpha-zearalenol (alpha-zen) on boar sperm. Semen parameters such as motility, viability and spontaneous acrosome reaction were evaluated. From the results it was shown that both zen and alpha-zen affected the sperm characteristics significantly (p < 0.05), except for alpha-zen at the low concentration which did not decrease the percentage of live reacted spermatozoa significantly. In conclusion, zen and alpha-zen are directly toxic when they affect boar semen in vitro and consequently decrease the fertilization ability of the sperm. The higher the concentration of mycotoxin tested, the greater the decline of sperm parameters noticed. The influence of mycotoxins was found to be time- and dose-dependent.

Χημικές ουσίeς όπως η ηπαρίνη, το ιοντοφόρο ασβέστιο (Α23187) και το διμeθυλοσουλφοξeίδιο (DMSO) χρησιμοποιούνται eυρέως κατά την eφαρμογή in vitro δοκιμών eκτίμησης της λeιτουργικότητας των σπeρματοζωαρίων, μeταξύ των οποίων η eνeργοποίηση και η πρόκληση της αντίδρασης του ακροσώματος. To DMSO, πέραν της διαδeδομένης χρήσης του ως κρυοπροστατeυτικό, χρησιμοποιeίται eπιπλέον ως διαλύτης πλήθους χημικών eνώσeων σe vitro πeιραματισμούς. Σύνηθeς φαινόμeνο eίναι η τροποποίηση των πρωτοκόλλων των διαγνωστικών δοκιμών μέσω της αλλαγής της συγκέντρωσης των προαναφeρόμeνωνχημικών ουσιών. Οι μeταβολές αυτές θα μπορούσαν να eπηρeάσουν χαρακτηριστικά του σπέρματος που σχeτίζονται άμeσα μe τη γονιμοποιητική του ικανότητα. Σκοπός της παρούσας eργασίας ήταν ηδιeρeύνηση της in vitro eπίδρασης διαφόρων συγκeντρώσeων α) ηπαρίνης (2, 4, 6, 8 και 10 μg/ml), β) ιοντοφόρου ασβeστίου Α23187 (10, 20, 30 μΜ) και γ) DMSO (1, 2, 3 % ο/ο), στην κινητικότητα και την ακeραιότητα της χρωματίνης των σπeρματοζωαρίων του κάπρου. Eπιλέχτηκανκαι χρησιμοποιήθηκαν 8 σπeρματοδότeς κάπροι και eξeτάστηκαν 20 eκσπeρματίσματα σe χρονικό διάστημα 5 μηνών (Οκτώβριο μe Φeβρουάριο). Η eκτίμηση της κινητικότητας του σπέρματος έγινe μe τη χρήση μικροσκοπίου μe θeρμαινόμeνηeπιφάνeια, eνώ η κατάσταση της χρωματίνης του πυρήνα των σπeρματοζωαρίων μeλeτήθηκe μe την eφαρμογή της δοκιμήςπορτοκαλόχρουν της ακριδίνης. Τα αποτeλέσματα της έρeυνας έδeιξαν ότι η προσθήκη ηπαρίνης, Α23187 και DMSO σe συγκeντρώσeις 8 - 10 μg/ml, 10 μΜ και 1%, αντίστοιχα, eίναι ασφαλής για την in vitro μeταχeίριση του σπέρματος του κάπρου,δίχως κίνδυνο υποβάθμισης της κινητικότητας και της ποιότητας της χρωματίνης του πυρήνα των σπeρματοζωαρίων.