HOS Test Research Articles

Comparison of different semen extenders and cryoprotectant agents to enhance cryopreservation of rabbit spermatozoa

The purpose of this research was to find a suitable protocol to enhance frozen rabbit sperm preservation analysing the role that seminal plasma (SP) plays and the effect of different cryoprotectant agents on sperm quality 0 and 2 h after thawing. Sperm samples were pooled and divided in eight fractions. Four of them were diluted with BotuCrio<sup>®</sup> (extender A), INRA 96<sup>®</sup> plus 6% glycerol (extender B), 6% N, N-dimethylformamide (extender C) and 6% N-methyl-2-pyrrolidone (extender D), respectively. The other four fractions were centrifuged and the supernatant was discarded in order to eliminate SP. Each sample was then resuspended with extender A, B, C and D. Samples were cooled progressively, loaded into 0.5 ml freezing straws and frozen with liquid nitrogen vapour. Thawing was performed by placing the straws into a bain-marie at 37°C for 21 s. Straws were dried and sperm samples placed into Eppendorf tubes to be analyzed by ISAS software, vitality test, HOS test and acrosome integrity test. The best motility and velocity parameters were obtained by extender A (P < 0.050) even when the motility parameter was compared with previous studies using other diluents. Additionally, sperm quality decreased over incubation time (P < 0.050) and no differences were found in samples processed with or without SP. This research revealed that BotuCrio<sup>®</sup> could be used for rabbit sperm cryopreservation and moreover the improvement of the cryopreservation process of rabbit sperm due to the demonstration that SP removing is not required.

Deoxynivalenol reduces quality parameters and increases DNA damage in mice spermatozoa.

This study was performed to investigate in vitro effects of deoxynivalenol (DON) on mice sperm quality parameters including viability, motility and DNA damages at various concentrations and exposure times. Mice spermatozoa were exposed to DON at 0, 2.5, 5 and 10µM for 1, 3 and 6hr, motility parameters were evaluated by computer-assisted analysis and viability was examined by colorimetric metabolic activity assay and HOS test. DNA damage was examined by acridine orange staining, and sperm damages via lipid peroxidation pathway were determined by malondialdehyde (MDA) content measurement. DON affected sperm parameters in a concentration- and time-dependent manner. In all test groups, the average path velocity and progressive motile spermatozoa were remarkably reduced. In comparison with the controls, after 1, 3 and 6hr exposure to DON, viability of spermatozoa was reduced 25, 30 and 49% respectively. DON exposure at 10µM for 6hr resulted in 15% DNA damage and 2.5-fold more MDA generation, when compared with nonexposed spermatozoa. Our data suggest that DON causes sperm quality parameters decline in concentration- and time-dependent fashion, which attribute to the reduction in sperm metabolic activity and membrane integrity and equally to increase in lipid peroxidation rate and DNA damage.

A preliminary study on the use of jenny colostrum to improve quality in extenders for freezing donkey semen

Sperm quality in donkeys (Equus asinus) after freezing thawing is still considered lower than that from other animals, including horses. The aim of this study was to test a new freezing extender supplemented with jenny colostrum on donkey sperm. After thawing, we evaluated sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodide (PI), membrane functional integrity by HOS-test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC-PNA) and PI. Ejaculates were collected from five fertile Donkeys. Sperm samples were pooled, diluted and cryopreserved into three experimental extender groups: BotuCrio®, lactose-extender supplemented with egg yolk (20%) and lactose-extender supplemented with jenny colostrum (20%). The results demonstrated that lactose-jenny colostrum samples displayed significantly higher values in almost all parameters evaluated (p < 0.05) compared with the other two extenders after thawing (BotuCrio® and lactose-egg yolk based extender, respectively) –Total Motility, Viability, HOS test, VCL, VSL and VAP. Acrosome status, LIN, STR and WOB despite showing lower values, none of them were statistically significant (p > 0.05). In conclusion, the extender containing jenny colostrum can be successfully used for donkey semen crypreservation and could effectively improve donkey sperm qualities after freezing -thawing.

Effect of glycerol, n, n-dimethylformamide and n-methyl-2-pyrrolidone on rabbit sperm stored at 4 °C and 16 °C

Artificial insemination with cooled semen is the most common practice in rabbit farms and any improvement on it helps to increase the efficiency and productivity of rabbit meat farms. Therefore, the aim of this study was to assess whether different cryoprotectant agents (CPA) as glycerol, N, N-Dimethylformamide (DMF) and N-Methyl-2-Pyrrolidone (NMP) can improve cooled rabbit sperm quality stored at 4ºC and 16ºC. Sperm samples were diluted with INRA 96® (Extender A), INRA 96® with 6% glycerol (Extender B) or 6% DMF (Extender C) or 6% NMP (Extender D) respectively and stored at 4ºC and 16ºC. Samples were then analysed at 4, 24, 48 and 72 hours after refrigeration by integrated sperm analysis system (ISAS®), eosin-nigrosin stain (vitality), hypo-osmotic swelling test (HOS test) and acrosome integrity test. Extender C showed higher percentage of motility, vitality and HOS test than extender B and D (p<0.05). Whereas sperm quality decreased over time (p<0.05), data showed that the addition of DMF kept the motility and sperm plasma membrane integrity after 24 hours of storage better than other diluents. These results suggest that the addition of DMF to INRA 96® exerts a protective effect on the membrane of spermatozoa improving seminal quality.

Improvement of post-thawed sperm quality in broiler breeder roosters by ellagic acid-loaded liposomes

ABSTRACTLiposomes could improve the delivery of substances to sperm. This study was conducted to investigate the effect of the antioxidant ellagic acid and ellagic acid-loaded liposomes on post-thawed sperm quality in broiler breeder roosters. Semen was diluted in Beltsville extender containing ellagic acid or ellagic acid-loaded liposomes (ellagic acid at 0 (control), 0.5, 1, and 2 mM) and cryopreserved. Sperm quality was evaluated post-thawing: motility characteristics (Computer-Assisted Semen Analysis), membrane functionality (HOS test), abnormal morphology, mitochondrial activity (Rhodamine 123), apoptotic status (Annexin V/Propidium iodide), malondialdehyde, and antioxidant activities (glutathione peroxidase (GPx), superoxide dismutase (SOD), and total antioxidant capacity (TAC)). The results showed that 1 mM ellagic acid-loaded liposomes improved total motility, membrane functionality, and viability comparing to 0.5 and 2 mM ellagic acid, 2 mM ellagic acid-loaded liposomes, and control group. Mitochondrial activity was significantly higher for 1 mM ellagic acid-loaded liposomes compared to the rest of the treatments, except 1 mM ellagic acid. Ellagic acid at 1 mM in both forms significantly increased GPx and TAC after freeze-thawing (no significant variation for SOD), and also yielded the lower proportion of apoptotic and dead cells. In conclusion, ellagic acid improved post-thawed sperm quality in broiler breeder roosters. The use of liposomes could further enhance the effects of ellagic acid.

Influence of idebenone on ram semen quality stored at 4°C.

The current study was designed to investigate the effect of idebenone (Id), an antioxidant on ram semen quality. Semen samples were collected, pooled and diluted in a Tris-based extender supplemented with 0, 1, 2, 4 and 8µM idebenone. Computer-assisted sperm analysis was used to evaluate spermatozoa kinematics. Sperm viability and membrane functionality were assessed respectively, by eosin-nigrosin staining and HOS test. Biochemical assays were carried out to measure different metabolites in spermatozoa and medium at 0, 24, 48 and 72hr. Total and forward progressive motility were greater in 1, 2 and 4µM idebenone treated groups compared to control at 24, 48 and 72hr time points (p<0.05). Semen supplementation with Id significantly increased viability and functionality of spermatozoa membrane during storage (p<0.05). Lower amounts of lipid hydroperoxides in medium and spermatozoa were observed in Id-treated groups compared to control one at 24 and 48hr of storage (p<0.05). Medium and spermatozoa amounts of malondialdehyde and nitric oxide were less in Id 4µM group compared to the control at 72hr (p<0.05). Total antioxidant capacity values and superoxide dismutase activity of spermatozoa and medium were greater in 2 and 4µM idebenone treated groups in comparison with the control at 72hr (p<0.05). Results of the current study indicated that ram semen supplementation with Id at 4µM level improved quality by ameliorating nitrosative and peroxidative stress, hence could be considered as an antioxidant additive during storage at 4°C.

Extender osmolality, glycerol and egg yolk on the cryopreservation of epididymal spermatozoa for gamete banking of the Cantabric Chamois (Rupicapra pyrenaica parva)

Germplasm banking is a key technology enabling the ex-situ conservation of wild species. However, cryopreservation protocols must be tested to assure the applicability of the banked material. The objective of this study was defining a range of parameters for the composition of a semen extender for Cantabrian chamois epididymal spermatozoa (post-mortem collection). The freezing extender was based in a TES-Tris-fructose buffer, modifying its composition in three experiments: Osmolality of the buffer (320, 380 or 430 mOsm/kg, 8% glycerol, 15% egg yolk), glycerol (4 or 8%, 430 mOsm/kg, 15% egg yolk), egg yolk (10 or 15%, 430 mOsm/kg, 4% glycerol). Sperm was extended at 100 mill. spermatozoa/ml, cooled at 5 °C and frozen at −20 °C/min. Sperm quality was assessed pre and post-thawing (CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). Freezability was good overall, with total motility of 65.5% ± 2.4 initial and 55.8% ± 2.4 post-thawing. The extenders affected the post-thaw sperm quality marginally. Whereas osmolalities and glycerol concentrations seemed not to differ, 430 mOsm/kg and 4% glycerol might be preferred. Egg yolk concentrations only differed on sperm velocity (VCL: 84.0 ± 6.7 μm/s in 10% vs. 70.7 ± 6.2 μm/s in 15%, P < 0.05). Our results suggest a good cryotolerance of chamois epididymal spermatozoa, with a preferred extender composition of hyperosmotic buffer, glycerol in the 4% range and lower egg yolk (10% range) than other ruminants.

Use of aqueous extract of noni in extender for sheep semen freezing

ABSTRACT The study aimed to evaluate the action of aqueous extract of noni in an extender for sheep semen freezing. Treatments differed in inclusion of aqueous extract of noni in the extender: T1 ˗ no addition; T2 ˗ 24µg/mL; T3 ˗ 72µg/mL; and T4 ˗ 120µg/mL. Ejaculates were collected, diluted in the four treatments, and frozen. After thawing, the semen was subjected to a thermoresistance test and evaluated for subjective motility, vigor, membrane integrity assessment by hypo-osmotic swelling test, live-dead assay, computer-assisted sperm analysis and the status of sperm capacitation and acrosome reaction. Data were subjected to ANOVA, and then to Student Newman Keuls’s test at 5% significance level. In the thermoresistance test after two hours of incubation, motility in T4 (120µg/mL) was lower than in the other treatments, with no differences in the HoS test in either diluted semen or in the semen evaluated immediately post-thawing, while for the other times, treatments showed similar responses. Regarding the motility parameters, a difference was observed for progressive motility, curvilinear velocity, average path velocity, and amplitude of lateral head displacement. As to the sperm capacitation status, a difference was observed between treatments for the sperm capacitated with intact acrosome.

Effect of length of time post-mortem on quality and freezing capacity of Cantabric chamois (Rupicapra pyrenaica parva) epididymal spermatozoa

Genome Resource Banks are keystones in the ex-situ conservation of wild species. Post-mortem (PM) collection of epididymal spermatozoa is an opportunistic and valuable source of germplasm, the time from the death of the animal limits its use. Seeking to improve germplasm preservation strategies for the chamois (Rupicapra sp.), the effect of PM time on epididymal sperm quality and freezability was studied using the Cantabrian chamois. Samples were classified according to PM collection time, up to 216 h (refrigerated), and cryopreserved (Tris-citric acid-fructose, 430 mOsm/kg, 15% egg yolk, 8% glycerol; freezing at −20 °C/min). Sperm quality was assessed after recovery and post-thawing (motility by CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). The sperm mass pH and osmolality showed a positive correlation with time. Total sperm motility dropped after 2 days PM, with progressivity and sperm velocities remained similar up to 3 days PM. Sperm freezability was acceptable, with the post-thawing HOST, motility, progressivity, VAP, VCL, VSL and BCF negatively correlating with PM time. Overall, chamois epidydimal samples were not adequate for preservation after 6 days PM. Freezability capacity could make these spermatozoa suitable for specific ART even if kept refrigerated for several days PM.

Effect of thawing method on bull sperm survival in ejaculates frozen in 4 ml and 8 ml volumes

The aim of this study was to evaluate the effect of different thawing protocols (slow (P1), medium (P2), and fast (P3)) on percentage of motile sperm (MOT) and percentage of sperm cells with intact membranes (INT) in Holstein (4 bulls; 72 samples) and Czech Fleckvieh (4 bulls; 72 samples) semen frozen-thawed in 4 ml and 8 ml volumes. MOT was analysed in fresh semen, as well as immediately after thawing (T0) and 30 min after thawing (T30). INT was analysed using hypoosmotic swelling test (HOS test) in fresh ejaculate (FE), after diluting (DE), and at T0. The differences between FE parameters and frozen-thawed ejaculate parameters, expressing changes that occur during cryopreservation, were calculated. Apart from the effect of thawing protocol, the effect of breed and the effect of quality of FE expressed by MOT immediately after collecting were evaluated, too. Unlike thawing of semen cryopreserved in straws (0.25 and 0.5 ml), thawing using the slow protocol (P1) was the most appropriate (P &amp;lt; 0.05) for both observed volumes. There were found significantly higher MOT in the volume of 8 ml in both T0 and T30 and in the volume of 4 ml in T30 in samples thawed using P1 and P2. MOT in T0 was significantly affected by breed in samples frozen in 8 ml and in T30 in samples frozen in 4 and 8 ml. There were found no significant differences in INT in all reported volumes, however decrease of INT during cryopreservation was affected by breed.

Effect of Achillea millefolium-loaded nanophytosome in the post-thawing sperm quality and oxidative status of rooster semen

The aim of this study was to compare the effectiveness of antioxidants including Achillea millefolium extract (AmE) (n0t1.5: 1.5, n0t3: 3 and n0t4.5: 4.5 mg/L) and AmE loaded in nano phytosome (n1t1.5: 1.5, n1t3: 3 and n1t4.5: 4.5 mg/L) in the freezing of Ross 308 rooster semen. Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality and enzymatic parameters (SOD, CAT and GPx) were assessed after thawing. AmE-loaded nano phytosome at a concentration of 3 mg/l resulted in significantly (P < 0.05) higher total motility (MOT) (73.78 ± 2.92) and at concentrations of 1.5 mg/L and 3 mg/L in progressive motility (PROG) (14.12 ± 0.38, 16.78 ± 0.38) in comparison with the control group (MOT: 58.48 ± 2.92; PROG: 9.08 ± 0.38). Sperm viability (Vi) was higher (P < 0.05) in n1t3 (74.62 ± 1.55) and membrane integrity (Mi) in n0t3 and n1t3 groups (65.91 ± 1.91, 63.73 ± 1.91, respectively) compared to the control groups (Vi: 66.85 ± 1.55; Mi: 53.18 ± 1.91). Moreover, the lowest percentage of MDA was measured in n1t3 group (1.31 ± 0.31). There was no significant difference for SOD and CAT values with the use of various extenders.In conclusion, we suggest that AmE loaded in nano phytosome at 3 mg/l dose can be added to basic extender for improving rooster sperm motility, viability and oxidative stress values during the freezing procedure.

Effects of seminal plasma and different cryoprotectants on rabbit sperm preservation at 16°C

The purpose of this research was to assess whether the presence of seminal plasma (SP) can improve sperm quality of rabbit spermatozoa stored at 16°C for 72 h and moreover evaluate the cryoprotectant effects of glycerol, N-N-Dimethylformamide (DMF), and N-methyl-2-pyrrolidone (NMP). Semen samples were pooled and divided in eight fractions. Four of them were diluted with INRA (extender A), INRA with 6% glycerol (extender B), INRA with 6% DMF (extender C), or INRA with 6% NMP (extender D), respectively. The other four fractions were centrifuged, and the supernatant was removed in order to eliminate SP. Each sample was then resuspended with extender A, B, C, or D, respectively. All samples were stored at 16°C and analysed at 4, 24, 48, and 72 h by ISAS®, vitality test, HOS test, and acrosome integrity test. After analyse of the results, SP samples showed a significantly higher percentage (P=0.020) in the HOS test (71.9 ± 1.6%) than non-SP samples (66.5 ± 1.6%). Non-SP samples had better results for kinematic parameters. Extenders A and C showed great results for the percentage of motile spermatozoa (63.1 ± 4.3% and 63.4 ± 3.7%, respectively), vitality (88.9 ± 2.6% and 87.7 ± 2.7%, respectively), and HOS test (68.9 ± 1.4% and 75.2 ± 1.4%, respectively). Extenders B and D showed worse data for sperm quality. These results suggest that SP has a protective effect on rabbit sperm membranes and maintains better sperm motility. The addition of glycerol and NMP to INRA does not improve rabbit sperm quality; nevertheless, the DMF cryoprotectant exerts a protective effect on the membrane of spermatozoa, improving seminal quality during rabbit sperm preservation at 16°C.

Effect of foot and mouth disease vaccination on seminal traits in pure Holstein Friesian bulls

The present investigation was carried out to study the effect of foot and mouth disease vaccination on seminal traits of 30 Holstein Friesian (HF) bulls, 3-5 years old, maintained at BAIF Central Research Station, Uruli Kanchan, Pune, India. The study period was from August to September 2013 and semen quality traits were evaluated on each bull maintained under identical feeding and management regimes. A total of 180 semen ejaculates were collected with one ejaculate from each bull at 3 days interval in a week at 15 days before, 15 and 30 days after FMD vaccination. Raksha Ovac vaccine of Indian Immunologicals Ltd. was used for vaccination purpose. Seminal traits like fresh and post thaw sperm motility, sperm concentration, semen volume, live and dead count, plasma membrane integrity were evaluated for all the ejaculates. Data on seminal traits were analysed by using one way ANOVA. Results revealed that vaccination had significant (P less than 0.05) effect on post thaw motility and highly significant (P less than 0.01) deleterious effect on HOS test, head and mid piece abnormalities; however no effect was noted on ejaculate volume, sperm concentration, initial motility, viability and tail abnormalities. The observations showed that vaccination alters the seminal traits of pure HF bulls.

Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric, microscopic and oxidative parameters in ram semen

This investigation was carried out to study the effect of soybean lecithin 1.5% (wt/vol) (0, 2.5, 5 and 7.5 mg l−1 pomegranate extract (PE)) or PE-loaded lecithin nanoliposome (0, 2.5, 5 and 7.5 mg l−1) to Tris-based extender. Sperm motility (CASA), viability, membrane integrity (HOS test), abnormalities, mitochondrial activity, apoptosis status, lipid peroxidation, total antioxidant capacity (TAC)) and antioxidant activities (GPX, SOD) were investigated following freeze-thawing. No significant differences were detected in motility parameters, viability, membrane integrity, and mitochondria activity after thawing sperm between soybean lecithin and lecithin nanoliposomes. It was shown that PE5 significantly improved sperm total and progressive motility, membrane integrity, viability, mitochondria activity, TAC and reduced lipid peroxidation (malondialdehyde concentration). Moreover, the percentage of apoptotic sperm in PE5 extenders was significantly the lowest among other treatments. Sperm abnormalities, SOD and GPX were not affected by the antioxidant supplements. For apoptotic status, no differences were observed between soybean lecithin and lecithin nanoliposome. We showed that lecithin nanoliposome extender can be a beneficial alternative extender to protect ram sperm during cryopreservation without any adverse effects. It was also observed that regarding pomegranate concentration, PE5 can improve the quality of ram semen after thawing.

Spermicidal efficacy of VRP, a synthetic cationic antimicrobial peptide, inducing apoptosis and membrane disruption.

Presently available contraceptives are mostly hormonal or detergent in nature with numerous side effects like irritation, lesion, inflammation in vagina, alteration of body homeostasis, etc. Antimicrobial peptides with spermicidal activity but without adverse effects may be suitable alternatives. In the present study, spermicidal activity of a cationic antimicrobial peptide VRP on human spermatozoa has been elucidated. Progressive forward motility of human spermatozoa was instantly stopped after 100 μM VRP treatment and at 350 μM, all kinds of sperm motility ceased within 20 s as assessed by the Sander-Cramer assay. The spermicidal effect was confirmed by eosin-nigrosin assay and HOS test. VRP treatment (100 μM) in human spermatozoa induced both the intrinsic and extrinsic pathways of apoptosis. TUNEL assay showed VRP treatment significantly disrupted the DNA integrity and changed the mitochondrial membrane permeability as evident from MPTP assay. AFM and SEM results depicted ultra structural changes including disruption of the acrosomal cap and plasma membrane of the head and midpiece region after treatment with 350 μM VRP. MTT assay showed after treatments with 100 and 350 μM of VRP for 24 hr, a substantial amount of Lactobacillus acidophilus (about 90% and 75%, respectively) remained viable. Hence, VRP being a small synthetic peptide with antimicrobial and spermicidal activity but tolerable to normal vaginal microflora, may be a suitable target for elucidating its contraceptive potentiality.

Short communication: Progressive motility of frozen-thawed canine semen is highest five minutes after thawing.

Progressive motility is usually estimated by visual inspection using a light contrast microscope at X 100 immediately after semen collection or immediately after thawing frozen semen. Standard operating procedures have never been established for this test. The objective of this experiment was to examine time-dependent changes of motility after thawing cryopreserved canine semen. Semen of 35 dogs was collected, and volume, concentration, progressive motility, morphology, membrane integrity and HOS test were evaluated. For cryopreservation, CaniPRO® Freeze A&B was used. Semen was thawed and diluted using CaniPRO® culture medium. After thawing, semen was evaluated as before. In addition, every sample was evaluated for progressively motile sperm cells 0, 5, 20 and 60min after thawing. Progressive semen motility was significantly highest five minutes after thawing.

Ethylene glycol, but not DMSO, could replace glycerol inclusion in soybean lecithin-based extenders in ram sperm cryopreservation

The aim of this study was to evaluate the effects of glycerol, ethylene glycol or DMSO in a soybean lecithin extender for freezing ram semen. In this study, 20 ejaculates were collected from four Ghezel rams and diluted with soybean lecithin extender with glycerol (7%), ethylene glycol (3%, 5% and 7%) or DMSO (3%, 5% and 7%). Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality, mitochondrial activity (Rhodamine 123) and apoptotic features (Annexin V/Propidium iodide) were assessed after thawing. There was no significant difference between glycerol and ethylene glycol at different concentrations (3% and 5%) regarding sperm total and progressive motility, viability, and membrane integrity. The least percentages of mitochondrial functionality were observed in samples frozen with all different DMSO concentrations tested (P<0.05). Moreover, the percentage of post-thawed dead sperm was the greatest for all the DMSO concentrations compared with other groups (P<0.05). Thus, DMSO had an adverse effect on the post thaw ram sperm parameters. In contrast, ethylene glycol could be a desirable substitute of glycerol in the freezing extender, in view of similar results obtained in post-thaw quality of ram semen cryopreserved in a soybean lecithin extender. We propose that glycerol in a soybean lecithin based extender could be replaced by ethylene glycol at 3% or 5% concentrations.

43 CORRELATIONS OF METHODS OF SPERM ANALYSIS IN FRESH SEMEN OF SOUTH AFRICAN INDIGENOUS GOAT

This study investigated the correlations between methods of assessing sperm qualities; namely, sperm total motility (TM), sperm vitality, acrosome integrity, DNA integrity, and sperm membrane integrity. A total of 60 ejaculates from 6 bucks were collected and the spermatozoa evaluated. The overall mean percentages of sperm TM, sperm vitality (eosin-nigrosin and propidium iodide stains), sperm acrosome integrity (Spermac and SpermBlue® stains), sperm DNA integrity (acridine orange and halotech), and sperm membrane integrity (hypoosmotic swelling test and water test) were 94.7 ± 0.5, 81.0 ± 0.6. 79.6 ± 3.7, 79.7 ± 1.8, 78.6 ± 5.3, 75.7 ± 5.5, 74.3 ± 5.1, 73.1 ± 3.5, and 73.4 ± 3.6, respectively. There were significant correlations (P &lt; 0.05) between mean percentage live sperm evaluated with eosin-nigrosin stain and sperm TM (r = 0.813), between percentage intact acrosome assessed with SpermBlue® and sperm TM (r = 0.846), and between SpermBlue® and eosin-nigrosin (r = 0.965). There were highly significant correlations (P &lt; 0.01) between sperm membrane integrity evaluated with HOS test and sperm TM (r = 0.871), between percentage of intact sperm DNA assessed with halotech and SpermBlue® (r = 0.832), and between percentage of intact spermatozoa DNA assessed with acridine orange and percentage intact acrosome evaluated with spermac stain (r = 0.862). Under the conditions of this study, the correlated methods of sperm analysis proved suitable for analysis of goat spermatozoa and can serve as useful indicator of potential fertility for sperm. They could be used for accurate assessment of the individual sperm cell rather the population as a whole. Motility, eosin-nigrosin stain, SpermBlue®, halotech and acridine orange stain still remain practical and valuable tools for predicting sperm fertilizing ability.

Meningkatkan Kemampuan Generalisasi Matematis Melalui Discovery Learning dan Model Pembelajaran Peer Led Guided Inquiry

The low ability of mathematical generalization of the students of SMPN 17 Bandar Lampung is because most of the students still have difficulty in drawing a conclusion from the material they have acquired and the lesson learned process of generalization aspect or less precisely the instructional model used. The purpose of this research is to know the influence of learning model of Peer Led Guided Inquiry and learning, discovery learning model with the ability of mathematical generalization. The measured variable from this research is the generalization ability in mathematics learning with the subject of algebraic factorization. This research is a quasi experimental research (quasi experimental research), where the subject of the study is students Class VIII students SMPN 17 Bandar Lampung. Data collection technique used is a test of mathematical generalization ability. The data analysis technique used analysis of variance (ANAVA) of one road with cell unlike the significance level α = 0,05 and as requirement analysis, that is a normal distribution population with liliefors model and homogenous population with Bartlett Test. Based on the result of research using ANAVA one way with cell unequal with significance level α = 0,05 it is obtained that 〖F〗 _count F_tabel IE 7,5105 3,0973, hence the decision of Ho test is rejected. So it can be concluded that there is influence of learning model Peer Led Guided Inquiry, learning model of Discovery Learning, Conventional learning model to ability of mathematical generalization of learners. The test proceeds with a double-compression test using Scheffe ' methods to see which has a significant effect on mathematical generabilizability. The calculation results show that of the three treatments, the learning model of Discovery Learning which gives a significant influence on the ability of mathematical generalization.

Prognostic value of a pre-freeze hypo-osmotic swelling test on the post-thaw quality of dog semen

Throughout cryopreservation, sperm are exposed to major osmotic challenges. Only intact membranes of sperm cells are able to regulate these volumetric changes, which can be determined by the hypo-osmotic swelling test (HOS test). Correlations between the HOS test and conventional semen variables are inconsistent. Therefore, the objectives of this study were (1) to examine relationships between HOS test results and standard semen variables before freezing and after thawing and (2) to evaluate the prognostic value of the HOS assessments on post-thaw quality of dog semen. Semen of 35 dogs was collected and analyzed before freezing and after thawing following a 7-day freeze–thaw interval. Conventional semen variables such as sperm cell motility, membrane integrity morphology were evaluated and the HOS test was conducted with results from this test being recorded. In fresh semen the HOS test was positively correlated with progressive motility of sperm cells: r=0.52, sperm cell membrane integrity: r=0.50 and normal sperm cell morphology: r=0.46 (P<0.05). In frozen–thawed semen, the data obtained with the HOS test were positively correlated with progressive sperm cell motility: r=0.67 and membrane integrity: r=0.86 (P<0.05). The data obtained with the HOS test in fresh semen were positively correlated with sperm cell membrane integrity: r=0.50 normal sperm cell morphology: r=0.55 and data from the HOS test (r=0.43; P<0.05) with frozen–thawed semen. For the prediction of individual cryopreservation capacity, results from assessment of the fresh semen variables of good and poor semen quality were statistically compared. Based on these results, it is not possible to predict the quality of frozen–thawed dog semen using the HOS test.