Hypo-osmotic Swelling Test Research Articles

Individual and Combined In Vitro Effects of Deoxynivalenol and Zearalenone on Boar Semen.

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’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 μM and 62.8 μM 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.

The evaluation of lycopene and cysteamine supplementation effects on sperm and oxidative stress parameters during chilled storage of canine semen.

The aim of this study was to evaluate the effects of different concentrations of lycopene and cysteamine on characteristics of sperm, liquid peroxidation and enzymatic activities in seminal plasma of canine semen preserved at 5°C for 72hr. The semen samples were divided into eight aliquots: control, control sham (dimethyl sulfoxide 5%), lycopene groups (250, 500 and 750µg/ml) and cysteamine groups (2.5, 5 and 10mM). Motility, viability, membrane integrity, DNA integrity, total antioxidant capacity (TAC) and malondialdehyde (MDA) levels were evaluated. Progressive motility and total motility were higher with the 500 and 750µg/ml lycopene concentrations, respectively, compared to the control group and the cysteamine groups following 72hr of storage in the liquid storage. Motility characteristics, viability and hypo-osmotic swelling test (HOST) percentages were significantly improved in 500µg/ml lycopene compared to other groups. The 500 and 750µg/ml lycopene concentrations, respectively, showed significantly reduced percentages of spermatozoa with DNA integrity compared to the control group. The 500 and 750µg/ml lycopene concentrations, respectively, led to the significant decrease of MDA levels. The 500µg/ml lycopene enhanced TAC levels after 48 and 72hr that was not observed in other groups. In conclusion, the findings of this study showed that lycopene supplementation in canine semen extenders improved canine semen parameters and TAC levels and decreased MDA levels in the chilling process.

Insemination of sows with seminal doses prepared by a two-step hypothermic dilution does not impair the reproductive performance at farm.

Reproduction in swine is mostly carried out through artificial insemination (AI). For this purpose, AI studs collect the ejaculates, analyse the sperm quality, dilute and package to produce seminal doses and ship them to sow farms to carry out the AI. Temperature is controlled during the process to avoid sperm damage. Semen is diluted in the extender in a one-step or a two-step process where the second can be isothermic (approximately 32°C) or hypothermic (room temperature 21-22°C). Both techniques are currently performed, and the latter could reduce time and costs, but the literature available comparing the processes is scarce and presents discrepancies. To date, there are no studies about its impact in fertility. This study compared hypothermic two-step dilution (HTSD) and isothermic two-step dilution (ITSD) in laboratory and field trial to elucidate whether HTSD has any effect. Ejaculates from 72 boars in nine AI studs were split and processed with both techniques using a high-performance extender and evaluated in laboratory. Four farms inseminated 345 sows with samples of four of these AI studs, and their fertility and prolificacy were registered. Results show no significant differences between doses prepared by HTSD and ITSD technique, having no impact in laboratory results (percentage of motile sperm, short hypoosmotic swelling test (sHOST) and short osmotic resistance test (sORT), viable sperm, damaged acrosomes, sperm under early apoptosis, high mitochondrial membrane potential (p>.1), fertility (92.2% versus 94.1%, p=.45) or farrowing rate (15.8±0.3 versus 16.1±0.3 p=.46). In conclusion, our results suggest that HTSD of semen on extender could be safely implemented in AI studs under the conditions tested.

Heparin binding proteins in seminal plasma of breeding buffalo bulls and their relation with semen freezability and in vivo fertility

The present study was conducted to determine the concentrations of heparin binding proteins (HBP) in seminal fluid of breeding buffalo bulls and establish their relationship with frozen-thawed semen characteristics and fertility. Both fresh and frozen semen were collected from same ejaculate of 30 breeding buffalo bulls. Based on HBP concentrations in seminal plasma obtained from affinity chromatography, all the bulls were divided into 2 groups, viz. high fertility bulls (HFB, ≥ 6.5 mg/ml; n=15) and low fertility bulls (LFB, < 6.5 mg/ml; n=15) for further comparisons. The frozen-thawed semen of same ejaculate was evaluated for per cent CASA-based sperm kinetics, viability, Hypo-osmotic swelling test (HOST), acrosome integrity, in vitro acrosome reaction and first service conception rate (FSCR). The overall HBP concentrations were significantly higher in seminal plasma (7.31±0.14 vs 5.33±0.19 mg/ml) and fresh (0.66±0.03 vs 0.59±0.02 mg/109 sperms) sperm extracts of HFB than LFB. The frozen-thawed semen characteristics, viz. total motility, per cent acrosome reaction and FSCR were also significantly higher in bulls with high fertility than in lower ones. No significant difference was observed in sperm viability, HOST and acrosome integrity in the 2 groups of bulls. Total 12 HBP bands in range of 11 to 135 kDa were recorded on SDS-PAGE. Higher levels of HBP in seminal plasma of HFB might be responsible for their better semen quality and fertility.

Antioxidative Effect of Addiing Taurine and Leptin to Tris-Extenders on Freezing And Fertilizing Abilities of Buffalo-Semen

Current study aimed to examine possible acts of adding taurine or leptin to extender on frezability and fertiablity of frozen buffalo-semen. Five sexually mature buffalo male were used for semen collection (twice/week for 35 days) using artificial vagina. Mass motility of all collected ejaculates was ≥70%. The collected semen was pooled, allotted into 7 replicates, then extended in Tris-egg yolk without (control), with taurine and leptin (20 and 40 ng/ml for each), or two combination of taurine (10 and 20 ng/ml) and leptin (20 and 20 ng/ml). Semen was frozen in straws for 2 wk and thawed at 370C/30 seconds. In diluted, equilibrated and thawed semen, the live, individual motility and abnormal sperm percentages were determined, while the hypo-osmotic swelling test (curled tail) was examine in semen after thawing. In the thawed seminal plasma, enzyme activity was assayed. Fertilizing ability of each semen treatment was determined. Results showed that adding 20 ng leptin/ml or 40 ng taurine/ml enhanced (P<0.05) all spermatozoa parameters after dilution, equilibration and thawing versus control and other treatments. Activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were reduced (P<0.05) with all additives compared with control semen. Pregnancy rate for buffalo cows which inseminated with extended semen contain 20 ng leptin /ml, 40 ng taurine /ml, and control was 90, 80 and 70% (P<0.05). Thia study concluded that, addition of 20 ng leptin/ml or 40 ng taurine/ml in the extender of Tris for buffalo frozen-semen had positive impact on sperm function during cryopreservation and increased fertility of buffalo-bull spermatozoa.

Ostrich ejaculate characteristics and male libido around equinox and solstice dates.

The study evaluated the effect of time of the year in which changes in photoperiod occurs on ostrich semen characteristics and male libido. Semen was collected for 5days before, on and 5days after winter solstice (21 June 2016), spring equinox (22 September 2016), summer solstice (21 December 2016) and autumn equinox (20 March 2017) in the southern hemisphere. Semen was collected from 10 South African Black ostrich males (average age ± standard deviation; 4.5 ± 2.27years) using the dummy female. Semen volume, sperm concentration, total sperm per ejaculate, sperm motility traits, percentage of normal sperm, head and tail abnormalities and percentage of affected sperm in the hypo-osmotic swelling test (HOS) were evaluated. Male libido defined as the willingness of males to mount the dummy female was also recorded. Semen samples collected around summer solstice, spring and autumn equinox were higher in sperm concentration and sperm output compared with winter solstice (P < 0.05). Study periods did not influence semen volume, sperm motility traits, the percentage of normal sperm, head abnormalities and HOS. Tail abnormalities were higher around winter solstice than around spring equinox (P < 0.05). Male libido and the success of semen collection were significantly higher around spring equinox (P < 0.05). Changes in photoperiod in the southern hemisphere do not affect semen production in ostriches. However, high sperm output and male libido around spring equinox and summer solstice dates suggest that these periods may be preferred for semen collection for artificial insemination and storage purposes.

Effect of Dietary Supplementation of Manganese, Chromium and Cobalt on Semen Qualities in Sahiwal Bulls

The present study was conducted to evaluate the effect of supplemental manganese (Mn), chromium (Cr) and cobalt (Co) on semen qualities in Sahiwal bulls. Twelve Sahiwal bulls divided into three groups T1, T2 and T3 based on their similar semen volume and concentration and were fed with 0, 1.5 and 2.0 times higher Mn, Cr and Co over and above the basal diet (ICAR, 2013) in T1, T2 and T3, respectively, for a period of 120 days. Semen was collected in all the groups twice a week and evaluated for quantitative and qualitative attributes. It was found that additional supplementation of Mn, Cr and Co had no influence on body weight gain, intake and digestibility of dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fibre (NDF) and acid detergent fibre (ADF)]. Seminal mass motility, initial progressive motility, live spermatozoa, normal spermatozoa, intact acrosome and hypo-osmotic swelling test (HOST) positive sperm count was higher (P less than 0.05) in T3 group in comparison to T1 and T2 groups of fresh as well as frozen semen. Blood superoxide dismutase (SOD) activity was higher (P greator than 0.05) in both T2 and T3 than T1. A reduction (P less than 0.05) in lipid peroxidation activity (LPO) in fresh and frozen semen in T3 group was observed. It may be concluded that dietary supplementation of Mn @ 30 ppm, Co @ 0.22 ppm and Cr @ 2 ppm, respectively per day per Sahiwal bull can improve the qualitative and quantitative attributes of semen.

Effects of reduced glutathione on ram sperm parameters, antioxidant status, mitochondrial activity and the abundance of hexose transporters during liquid storage at 5 ℃

The objective of this study was to evaluate the effects of reduced glutathione (GSH) supplementation on sperm motility, kinematics, mitochondrial activity and the abundance of sperm hexose transporters during liquid storage of ram semen at 5 ℃. Semen samples from eight rams were pooled and diluted with Tris-egg yolk extender supplemented with 0 (control), 50, 100 and 200 mM GSH, and they were assessed at different processing periods (fresh diluted, stored for 24, 48 and 72 h at 5 ℃). Sperm motility and kinematic parameters were analyzed by computer-assisted sperm analysis (CASA). The hypo-osmotic swelling test (HOST) was used to evaluate the functional integrity of sperm membrane. Eosin-nigrosin and JC-1/PI staining methods were used to assess sperm viability and mitochondrial activity, respectively. The antioxidant status of sperm was examined using biological assays. The changes of sperm hexose transporters (GLUT 3, 5 and 8) were determined using western blot analysis. As compared to the control group, supplementation with 100 and 200 mM GSH improved (P ＜ 0.05) sperm total motility, membrane integrity, motion parameters and mitochondrial activity at 72 h of storage, as well as resulted in the higher sperm antioxidant status after storage from 24 to 72 h. The cooling induced a reduction of GLUT 3 and 8, and GSH treatment at 200 mM retarded the gradual decline of these proteins during the prolonged storage time. Our data suggest that GSH can reduce the decline of semen quality by influencing the antioxidant status and energy metabolism of liquid stored ram spermatozoa. The concentration of 200 mM could be appropriate for ram semen during liquid storage at 5 ℃.

The Effect of Genistein on the Plasma Membrane Integrity of Frozen Ongole Grade Bull Semen Based on Skim Milk – Soy Lecithin Extender

Beef cattle represent livestock commodities that can help support national economic stability. Ongole grade bull (PO) is one of the local cattle breeds whose population and genetic quality decrease. Various efforts have been made in improving the genetic quality and in increasing the population of the local livestock population to achieve self-sufficient status of the beef supply in Indonesia. One of the efforts is artificial insemination (IB). The study aims at examining the effect of different concentrations of genistein semen extender on the post-thawing membrane integrity of Ongole grade bull semen. It is useful in developing the genistein as a protective agent against reactive oxygen species (ROS) during cryopreservation process. It was conducted at BPBPTDK, Center for Animal Breeding Development, Animal Feed, and Veterinary Diagnostics, in the Special Region of Yogyakarta from February 7th, 2019 to May 7th, 2019. The semen of two healthy Ongole grade bulls was consecutively collected five times in the period of 3 months using artificial vagina. The semen was cryopreserved using skim milk-soy lecithin semen based extenders at different concentrations of genistein, which were group A (1.0 mmol genistein), B (2.0 mmol genistein) and K (without any genistein as control). The observed parameters included individual spermatozoa motility, percentage of spermatozoa viability that was observed using Eosin nigrosine staining method and spermatozoa membrane integrity that was observed using the hypo osmotic swelling test (HOST) method. The observations were made on fresh semen, extended semen, and post-thawing semen. The results of the observation were then analyzed descriptively and statistically using regression correlation test and one-way analysis of variance (ANOVA). The results of the analysis showed that the addition of Genistein at the different concentrations of 1 mmol and 2 mmol resulted in higher motility, viability, and membrane integrity than the control and had significant effect (p<0.05) on the motility, the membrane integrity, and the acrosome integrity of the Ongole grade bull semen. The genistein concentration of 2.0 mmol gave the highest mean percentage of spermatozoa motility (57.50 ± 1.00 %) after freezing. The genistein concentration of 1.0 mmol gave the highest mean percentage of spermatozoa membrane integrity and acrosome integrity (52.45 ± 0.85 % and 49.58 ± 0.98 %) after freezing.

Does Coenzyme Q10 Exert Antioxidant Effect on Frozen Equine Sperm?

During semen cryopreservation, the sensitivity of equine sperm to oxidative stress is increased by the eliminated seminal plasma. Thus, antioxidant addition to the semen extender can be helpful to the sperm survival after freezing and thawing. This work aimed to test whether coenzyme Q10 (CoQ10) added in different concentrations to the INRA 82 freezing extender has antioxidant function on equine sperm to improve its fertilizing ability. Semen samples from five stallions were frozen with the extenders: (T1) INRA 82, control, (T2) T1+ 5 μM CoQ10, (T3) T1+ 25 μM CoQ10, and (T4) T1+ 50 μM CoQ10. After sample thawing, sperm motility and kinetics characteristics were evaluated using a computer-assisted sperm analysis and sperm membrane functionality and integrity were evaluated with a hypo-osmotic swelling test and an epifluorescence microscopy, respectively. The nitrite (NO2-) and hydrogen peroxide (H2O2) concentrations of the semen samples were measured with spectrophotometry. There was no difference on the sperm characteristics among all treatments (P > .05). However, the 25 μM CoQ10 (T3) decreased NO2− concentration (6.7 ± 2.2 μM/μg protein) compared with the treatments T1, T2, and T4 (64.3 ± 3.7, 59.4 ± 5.3, 45.1 ± 8.6 μM/μg protein), respectively, as well H2O2 concentration (1.8 ± 0.3 μM/μg protein) compared with the control (4.6 ± 0.4 μM/μg protein) and 5 μM CoQ10 treatments (4.8 ± 0.2 μM/μg protein, P < .05). In conclusion, 25 μM CoQ10 plays a significant role as antioxidant to the frozen equine sperm, decreasing NO2− and H2O2 concentrations. Thus, its addition to the INRA 82 freezing extender may be beneficial to the fertilizing ability of equine semen.

Dietary omega-3 fatty acids from linseed oil improve quality of post-thaw but not fresh sperm in Holstein bulls

Docosahexaenoic acid (DHA), a member of the n-3 fatty acid family present in fish oil, has several positive effects on bovine sperm, including membrane integrity, motility and viability, as well as cold sensitivity. Our objective was to investigate effects of varying amounts of omega-3 fatty acids from linseed oil, administered orally, on quality of fresh and frozen-thawed bull sperm. Twenty fertile Holstein bulls (874 ± 45.38 kg) were randomly and equally assigned to four groups and received encapsulated (rumen-protected) fats for 12 weeks, as follows: group P, 300 g palm oil; group Pl, 200 g palm oil + 100 g linseed oil; group pL, 100 g palm oil + 200 g linseed oil; and group L, 300 g linseed oil. Sperm quality of fresh and frozen-thawed semen was evaluated by routine assays including sperm motion characteristics (CASA), membrane integrity (eosin-nigrosin), membrane activity (hypo-osmotic swelling test; HOST) and malondialdehyde (MDA) content. There were no significant differences among groups in semen volume, sperm concentration or sperm quality parameters in fresh semen. However, after freezing-thawing, total and progressive motility in group P (59.61 ± 1.95 and 40.19 ± 2.48%, respectively; LSM ± SEM) were lower (P < 0.05) than in groups Pl (66.06 ± 1.95 and 47.53 ± 2.48%), pL (65.67 ± 1.95 and 47.48 ± 2.48%) and L (65.36 ± 1.95 and 47.62 ± 2.48)%, with no significant differences among the latter three groups. Furthermore, membrane integrity (eosin-nigrosin) and activity (HOST) were lower (P < 0.05) in group P (55.79 ± 2.15 and 42.19 ± 2.17%) compared to groups Pl (62.73 ± 2.15 and 48.93 ± 2.17%), pL (64.06 ± 2.15 and 50.01 ± 2.17%) and L (64.47 ± 2.15 and 49.68 ± 2.17%), with no significant differences among the latter three. Furthermore, there were more (P < 0.05) morphologically abnormal sperm in group P (25.99 ± 1.62%) than in groups Pl, PL and L (21.55 ± 1.62, 21.69 ± 1.62 and 20.90 ± 1.62%). In conclusion, feeding Holstein bulls 100–300 g linseed oil daily improved sperm cryotolerance.

Effect of astaxanthin nanoparticles in protecting the post-thawing quality of rooster sperm challenged by cadmium administration

The protective role of astaxanthin nanoparticles (Ast NPs, 25 mg/kg p.o) against cadmium (Cd, 1 mg/100 g b.w. SC), a known inductor of lipid peroxidation and changes in the antioxidant defense system in the Ross 308 breeder roosters sperm, was examined. Sperm motility (computer-assisted sperm motility analysis), membrane integrity (hypoosmotic swelling test), viability, total abnormality, and enzymatic parameters were assessed after thawing. The testis/body weight (mg/kg) ratio and HE staining results of testis were also performed. The obtained results showed that Cd induced detrimental effects on testis and sperm, while Cd treated by Ast NPs (Cd Ast) diminished this change compared to the Cd group. Cd-treated group resulted in significantly (P < 0.05) lowest total (37.29 ± 2.46) and progressive (5.84 ± 0.47) motility and decreased antioxidant enzyme activity (CAT, TAC, and GPx), as well as producing a significant (P < 0.05) decrease in testis weight (mg) compared to the control group. Treatment with Ast NPs (Ast NPs + Cd) had reversed Cd-induced changes in the antioxidant defense system and significantly prevented Cd-induced testis damage. In conclusion, the results of our work suggest that Ast NPs at 25 mg/kg act as a potent antioxidant in protecting rooster testes against oxidative stress induced by Cd.

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos.

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P<0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.

Genetic and non-genetic factors affecting semen production and quality characteristics of Gir cattle breed under semi-arid climate.

Aim:This study aims to evaluate genetic and non-genetic factors influencing semen production potential of Gir bulls.Materials and Methods:Data on semen quantity (n=6911) and quality (n=466) available from January 2011 to December 2018 at BAIF’s frozen semen station, Jind, Haryana, India, were utilized for the study. Factors, namely, season of collection, age at collection, and bull (random effect) were studied for their effect on quantitative and qualitative semen traits. Least square means for the traits were obtained using a general linear model. The effect of age within bull for repeatable traits was analyzed using a longitudinal model with age as the control variable. Multivariate analysis using mixed repeatability model equation was utilized to estimate bull effect correlation (genetic + permanent environmental correlation), phenotypic correlations, and repeatability.Results:The overall least square means of ejaculate volume, sperm concentration, total sperms, initial and post-thaw motility, hypo-osmotic swelling test, and acrosome integrity of frozen semen were 6.62±0.03 ml, 1.22±0.01 109/ml, 8.09±0.05 109/ejaculate, 75.78±0.001%, 55.92±0.0001%, 55.13±0.005%, and 71.08±0.001%, respectively. The season of the collection showed a significant effect on volume, concentration, total sperm, and initial motility. The performance of bulls was superior in summer season, followed by winter and monsoon. Increase in semen attributes during summer season was due to the effect of lower temperature on sensitive stages of spermatogenesis. Age at collection had a significant effect on all semen traits. Volume and total sperm count showed increasing trend while concentration showed a decreasing trend with an increase in age. Motility and quality traits did not show any particular pattern. Individual bulls showed differences in all the semen performance traits with age. The repeatability of the traits ranged from 0.04 (HOST) to 0.58 (acrosome integrity). Bull effect correlation ranged from −0.73 (initial motility and acrosome integrity) to 0.93 (HOST and acrosome integrity).Conclusion:Individual bulls showed variation in traits measured over age. The result of the study could be utilized in suggesting suitable management plans to achieve the desired profit by improving semen quality in Gir bulls.

Influence of butylated hydroxytoluene addition to cryodiluents on freezability and DNA integrity of Boer and Zaraibi buck spermatozoa

Objective: To evaluate the cryoprotective effect of butylated hydroxytoluene on buck frozen semen. Methods: Semen was collected from Boer (n=6) and Zaraibi (n=6) bucks by electroejaculator for 5 weeks. Semen aliquots were diluted at 38 °C in Tris-buffer with egg yolk 15.0% (vol/vol) (Tris- egg yolk extender) or soya lecithin 2.5% (weight/vol) (Tris-soya lecithin extender) supplemented with butylated hydroxytoluene at 0.0 (as the control), 0.5, 1.0, 2.0 and 4.0 mM. Post-thawing motility (at 400x magnification), plasma (hypo-osmotic swelling test), acrosome (Trypan blue/Giemsa dual staining) membranes, DNA (comet assay), and lipid peroxidation (by malondialdehyde concentration) were assessed. Results: Spermatozoa motility was enhanced by butylated hydroxytoluene in Tris-soya lecithin extender at 0.5 mM in the two breeds, and in Tris-egg yolk extender at 1.0 mM in Boer and at 2.0 mM in Zaraibi bucks for up to 3 h post-thawing. Plasma and acrosome membranes and DNA integrity of the two breeds were maximally high with butylated hydroxytoluene at 1.0-2.0 mM in Tris-egg yolk extender and at 0.5-1.0 mM in Tris-soya lecithin extender. Lipid peroxidation was minimal with butylated hydroxytoluene at 1.0-2.0 mM in Tris-egg yolk and soya lecithin extenders in the two breeds. Butylated hydroxytoluene at 4.0 mM deteriorated spermatozoa motility, and plasma and acrosome membranes. Conclusions: The consequence of butylated hydroxytoluene on buck frozen-thawed spermatozoa varies with the levels of supplementation, buck breed, and phospholipid source in the extender. Semen parameters of Boer buck are better in their response to butylated hydroxytoluene than Zaraibi buck. Butylated hydroxytoluene at 1.0 and 2.0 mM in Tris-egg yolk extender, and at 0.5 mM in Tris-soya lecithin extender represents the best concentrations and profitably improves the semen quality of buck semen.

Relationship of frozen-thawed semen quality with the fertility rate after being distributed in the Brahman Cross Breeding Program.

Background and Aim:Various factors can reduce the quality of semen used for artificial insemination and have an impact on fertility decline, such as poor handling during frozen semen distribution. This study was aimed at assessing the quality of frozen-thawed semen after distribution in the field and its importance in maintaining fertility.Materials and Methods:The Brahman Cross (BX) breeding program of PT Lembu Jantan Perkasa, Indonesia, was used. This program was preferred due to its adherence to guidelines that limit the effects of extraneous factors that may affect semen quality. Frozen-thawed semen samples from eight bulls with the same production code were analyzed and compared between the production site (artificial insemination [AI] center) and the field (BX breeding program). Total and progressive motility (PM) of sperm were determined using computer-assisted semen analysis. Plasma membrane integrity (PMI) was assessed using hypoosmotic swelling test, sperm viability using Eosin-Nigrosin staining, acrosome integrity using trypan blue-Giemsa staining, morphological abnormalities using William staining, and DNA fragmentation using toluidine blue staining. The fertility rate was determined using the conception rate (%) derived from AI data based on 502 AI services and 478 cows in the BX breeding program. A t-test was used to compare the quality of frozen-thawed semen before and after distribution. The relationship between the qualities of frozen semen after distribution in the field with fertility was analyzed using Pearson correlation.Results:There was no significant difference (p>0.05) in the quality of frozen-thawed semen (sperm motility, PMI, viability, acrosome integrity, abnormalities, and DNA fragmentation) between the production site (AI center) and after distribution in the field (BX breeding program). The semen met the minimum standards for AI programs. Total motility (r=0.986), PM (r=0.961), sperm viability (r=0.971), PMI (r=0.986), and acrosome integrity (r=0.992) were all positively correlated (p<0.05) with fertility rate; while sperm abnormalities (r=−0.996) and sperm DNA fragmentation (r=0.975) were negatively correlated (p<0.05) with fertility rate.Conclusion:The study showed that to achieve the maximal and optimal fertility rate in bulls in an AI program, the overall quality of frozen-thawed semen in all aspects is critical. This can be achieved if the handling during distribution and storage, as well as the various factors that may affect the quality of semen in the field, can be controlled properly.

Purposeful cryopreservation of sperm prior to intrauterine insemination to overcome the embryo implantation defect associated with sperm with a subnormal hypoosmotic swelling test

Purposeful cryopreservation of sperm prior to intrauterine insemination to overcome the embryo implantation defect associated with sperm with a subnormal hypoosmotic swelling test

Effect of Caffeine and Pentoxifylline Added Before or After Cooling on Sperm Characteristics of Stallion Sperm

Different additives have been tested in cooled stallion sperm, in order to maintain sperm quality and to ameliorate the decrease in sperm fertility potential. In several species, caffeine and pentoxifylline promote sperm motility by increasing energy production. We evaluate the effects of caffeine and pentoxifylline when added to stallion sperm before or after cooling. Three ejaculates from five stallions each were processed and resuspended in skim milk extender. Caffeine (5 mM), pentoxifylline (3.5 mM), or both additives combined were included to sperm before or after cooling (4°C for 24 hours). Cooled sperm were incubated at 37°C and evaluated at 0, 30, 60, and 120 minutes for motility, morphology, viability (flow cytometry), and membrane functionality (hypo-osmotic swelling test). Results were analyzed by two-factor mixed model for repeated measures and Tukey comparisons. As main effects, the caffeine and pentoxifylline affected significantly motility and kinematic parameters, without interaction between treatment and incubation after cooling. No differences were observed whether the additives were added prior or after cooling. Pentoxifylline added after cooling reduced significantly motility during incubation, but with higher values at 30 minutes. We detected a decrease in morphologically normal sperm (P < .0001), caused by an increase of tail defects (P < .003) in the presence of both additives. Viability and membrane functionality were also significantly impaired by additives. Pentoxifylline when added after cooling improved sperm motility and kinematic parameters for a short period of time. However, sperm characteristic related to fertility potential was compromised after a prolonged exposure to caffeine or pentoxifylline.

159 Laparoscopic insemination method in sheep allows the use of an animal protein-free and inexpensive freezing medium

Animal-derived products are widely used in sperm cryopreservation for their cryoprotective properties. These components, however, must be replaced because of sanitary risks. STEMALPHA.CRYO3 (Ref. 5617, Stem Alpha), called CRYO3, is a chemically defined preservation medium currently used for freezing human tissue and adult stem cells. The aim of this study was to evaluate the effects of a CRYO3-based medium and of two cooling rates on invitro parameters and invivo fertility of ram sperm. Six rams (Blanche du Massif Central) were subjected to sperm collection four times using an artificial vagina. Sperm were split and frozen in three media: an egg yolk and milk-based medium (positive control), a CRYO3-based medium (tested medium), and a medium without additives (negative control). The two cooling rates were related to the distance between the straws and the surface of liquid nitrogen during the freezing process (5 and 20cm). Sperm membrane integrity (propidium iodide/SYBR-14), acrosome integrity (fluorescein isothiocyanate-peanut agglutinin/propidium iodide; FITC-PNA/PI), and mitochondrial membrane potential (JC-1) were assessed using flow cytometry, whereas functional membrane integrity was assessed using a hypo-osmotic swelling test and motion characteristics were evaluated using computer-assisted sperm analysis. Pregnancy rate, parturition rate, and prolificacy were evaluated after performing laparoscopic inseminations (n=75 ewes). Moreover, we characterised the freezing media thermodynamically using a differential scanning calorimeter. Statistical analyses were performed using R software. Invitro parameters were assessed using a mixed model including the time and the medium as fixed effects and the ram as a random effect. Pregnancy and parturition rates, following a binomial distribution, and prolificacy, assumed to follow a Poisson distribution, were analysed using generalised linear models, including the medium as a fixed effect and the ram as a random effect. Differences with P&amp;lt;0.05 were considered statistically significant. The cooling rates had no significant effect except on the wobble motion parameter. The positive control medium showed significantly higher results than the CRYO3-based medium and the negative control medium for all invitro parameters except for straightness motion parameter. Conversely, field trials showed no significant difference between the media for pregnancy rate (71, 64, and 74%), parturition rate (68, 61, and 74%) and prolificacy (2.0, 2.1, and 1.7), for the positive control, CRYO3-based medium, and the negative control, respectively. This study showed that the product, CRYO3, cannot replace egg yolk and milk in freezing extenders. Moreover, we showed that laparoscopic inseminations allowed a 74% parturition rate due to an easy and inexpensive medium comprising only a Tris buffer and glycerol. Although it could not be used on a large scale, this medium remains an option for international transport or long-term storage of genetic diversity.

26 Baobab oil supplemented extender preserves post-thaw bull sperm quality parameters

The processes of semen cryopreservation and thawing affect sperm membrane integrity and motility and increases morphological defects as well as DNA damage. The most influential cause of this is oxidative stress. When endogenous antioxidant capacity of seminal plasma is reduced during the freeze-thawing process, plant extracts exhibiting strong antioxidant activity can be used as supplements for compensation. Baobab oil has gained interest because it is rich in powerful antioxidants, which could protect sperm cells from oxidative damage during cryopreservation. Our study aimed to assess the effects of baobab oil on post-thaw sperm quality parameters in an egg-yolk-based extender. Thirty semen ejaculates were collected from 15 Nguni bulls using an electro ejaculator. Semen samples were randomly allocated to control (no baobab oil), 20μL (1%), 50μL (2.5%), and 100μL (5%) baobab oil per millilitre extender. Following dilution, semen samples were loaded into 0.25-mL semen straws, equilibrated for 4h at 5°C, and transferred into a controlled rate programmable freezer. The frozen semen straws were stored in a liquid nitrogen tank (−196°C) until thawing. Semen straws were thawed (37°C/60 minutes) after 1 week of cryopreservation and analysed for (1) sperm motility using a computer-aided sperm analyser, (2) morphological defects and viability using eosin-nigrosin stain, (3) membrane integrity by hypo-osmotic swelling test, and (4) DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. Data was analysed using analysis of variance. Treatment means were compared in relation to the control group by Dunnett's test. We found that supplementing semen extender with baobab oil at 1% significantly (P&amp;lt;0.05) preserved sperm DNA integrity (88.3±3.7) and membrane integrity (74.0±4.2) when compared with the control group (71.7±3.7 and 55.8±4.4, respectively). Baobab oil supplementation either at 1% (5.9±0.5), 2.5% (7.2±0.5), or 5% (6.0±0.5) significantly reduced sperm morphological defects compared with control (9.5±0.5). Total motility (1% (72.7%), 2.5% (72.7%), 5% (71.9%), control (59.3%)) and viability (1% (79.1%), 2.5% (79.8%), 5% (77.8%), control (67.6%)) were also improved by supplementation; however, the difference was not significant. In conclusion, it was demonstrated that supplementing bull semen extender with 1% baobab oil protects sperm from morphological defects, maintains membrane integrity, as well as preserves sperm DNA. All the baobab oil supplementation levels preserved post-thaw bull-sperm quality parameters.