Percentage Of Intact Acrosomes Research Articles

Effect of turmeric extract and dimethyl sulfoxide on buffalo semen freezability and fertility

The freeze-thaw process results in structural and functional damages caused by overaccumulation of reactive oxygen species (ROS). Addition of antioxidants to semen extenders is of a great importance to overcome this oxidative damage. The study objective was to evaluate the consequence of using Tris-citric acid fructose egg yolk (TCFY) extender supplemented with a combination of turmeric extract and dimethyl sulfoxide (DMSO) [TTD] on sperm freezability and fertility. From five tubes (each containing 5 mL TCFY), the first tube contained neither turmeric extract nor DMSO and was kept as control. The other four tubes contained 1.5% DMSO plus 100 µL/5 mL, 200 µL/5 mL, 300 µL/5 mL and 400 µL/5 mL turmeric extract. Semen samples were pooled and extended to reach an application of 60×106 sperm/mL (TT1D to TT4D, respectively). Diluted semen was exposed to the freezing protocol. The post cooling results revealed significant improvement in percent of alive spermatozoa in TT1D, significant decrease in sperm abnormalities in all concentrations used, significant improvement in intact acrosome percentage in TT1D, TT2D and TT4D. The post thawing results exhibited significant improvement in sperm motility in TT1D, TT2D and TT4D; significant amelioration of sperm membrane integrity (HOST) in TT1D, TT2D, TT3D and TT4D. Acrosome integrity was maintained in all concentrations as in the control. It was concluded that TT1D revealed the best semen quality in cooled semen; and TT1D, TT2D exhibited the superior post thawing semen quality. In addition, conception rate (CR) of the post-thawed semen was ameliorated in TT1D, TT2D, TT3D and TT4D with TT1D being the best one with this regard.

Effects of Calcium Nanoparticles on Male Rat Fertility and Sperm Function

Calcium is an essential regulating factor in a variety of biological functions including reproduction. It is widely required for different physiological activities in spermatozoa including spermatogenesis, sperm motility, capacitation, acrosome response, and fertilization. Recent advancements in nanotechnology have broadened its potential applications in biomedicine, including improving animal reproductive aspects. Our research was planned fundamentally to examine the influence of calcium deficiency and calcium administration using Nano and commercial calcium in two doses for each type of calcium (1000 mg/Kg body weight (BW) and 500 mg/Kg BW once daily for 64 days orally) to reveal their effects on male reproductive function as well as regulatory mechanisms connected to male fertility. Semen examination, biochemical analyses, enzymatic antioxidant, lipid peroxidation, and testis histopathology were all evaluated after 64 days. Our results revealed that Serum calcium, testosterone, and ABP levels, sperm count, motility, and percentage of intact acrosomes, as well as testicular antioxidant enzymes, were all considerably lower in the calcium-free diet group, whereas sperm abnormalities and testicular Malondialdehyde were significantly higher. In calcium (Nano and commercial) administered male rats, serum calcium, testosterone, and ABP levels, sperm count, motility, percentage of intact acrosomes, and testicular antioxidant enzymes all increased significantly, whereas sperm abnormalities and testicular Malondialdehyde dropped dramatically. Overall, these results point to a strong link between Ca2+, sperm function, and fertility outcomes. what's more, Ca2+ supplementation, particularly nanoparticles could efficiently improve male reproductive function and fertility.

Effect of fruit-juice on spermatozoa viability and lipid peroxidation of Red Sokoto bucks during liquid storage

Antioxidants are linked with sperm viability because of their protective effects against cell damage during preservation. In order to enhance the life span of refrigerated buck semen, this study was carried out to determine the effect of fruit-rich antioxidants on spermatozoa viability and lipid peroxidation (LPO) of buck semen during liquid storage. Pooled semen from five Red Sokoto bucks was diluted with Tris-egg yolk based extender and supplemented each with juices from pawpaw tomato and watermelon at 0, 2.5, 5, 7.5 and 10/ 100 ml respectively. Following dilution, the semen samples were assessed subjectively after in vitro storage at 5°C for 24, 48, 72 and 96 hours as regards sperm motility, abnormalities, and acrosome status using a phase-contrast microscope. The concentration of malondialdehyde (MDA) as indices of lipid peroxidation (LPO) in the stored semen was measured in thiobarbituric acid reactive substances (TBARS) at 24, 48, 72 and 96 hours. The results showed highest progressive motility in watermelon juice at 2.5% (P<0.05) during the first 24 hours of storage while the lowest progressive motility was recorded at various levels of pawpaw juice (P<0.05). After 48 hours of storage, extender supplemented with watermelon and tomato juices had better progressive motility compared to control except 7.5% and 10%% of tomato juice (P<0.05). Irrespective of level of juice in the extender, the percentage of intact acrosome was similar among the various juices and control. The results showed that spermatozoa extended with watermelon juice had the lowest (P<0.05) percentage abnormality compared to other extenders at 24, 48, 72 and 96 hours of storage. Higher (P<0.05) percent spermatozoa abnormality compared to other fruit juices and control was observed at 72 and 96 hours of storage in spermatozoa extended with pawpaw juice. Significant reductions of MDA concentrations were achieved by addition of fruit-rich antioxidants to Tris-egg yolk based extender during the first 72 hours and the reduction was much pronounced in extender supplemented with pawpaw juice compared to control (P<0.05). The findings reveal that fruit-rich antioxidants from watermelon and tomato have protective ability to maintain sperm viability and to reduce concentration MDA of buck semen during liquid storage.

Effect of bioactive peptide on ram semen cryopreservation

This present study investigated the effect of bioactive peptide (BAPT) (BAPT) on the quality of ram semen during cryopreservation. Ram ejaculates were extended with Tris buffer supplemented with no antioxidants (as control group), 20 μg/mL BAPT (as BAPT20 group), 40 μg/mL BAPT (as BAPT40 group) and 60 μg/mL BAPT (as BAPT60 group). After cryopreservation, sperm quality including motility, vitality, the percentage of hypoosmotic swelling test (HOST)-positive spermatozoa and the percentage of intact acrosomes was assessed. Furthermore, the malondialdehyde (MDA) in seminal plasma and spermatozoa were analyzed, followed by the measurement of superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px) levels in seminal plasma. After in vitro fertilization, the embryonic cleavage rates and development rates of different groups were analyzed to compare the developmental abilities of spermatozoa. The results showed that the post-thaw sperm motility was significantly higher in the BAPT60 group compared to those in the BAPT20, BAPT40 and control groups (P < 0.05). The percentage of live sperms significantly increased from 48.12 ± 2.35% for the BAPT20 group, 55.43 ± 2.16% for the BAPT40 group to 57.53 ± 3.15% for the BAPT60 group. The percentage of HOST-positive spermatozoa was significantly higher in the BAPT60 group than those in BAPT20, BAPT40 and control groups (P < 0.05). The MDA levels in seminal plasma and spermatozoa were significantly reduced with BAPT supplement (P < 0.05). Additionally, the SOD, CAT and GSH-Px levels in the BAPT experimental groups were significantly higher than those of the control group, which further indicated that BAPT significantly inhibit the reactive oxygen species (ROS) production during the cryopreservation of ram semen. Furthermore, the embryonic cleavage rates and development rates of the BAPT40 and BAPT60 groups were significantly increased in comparison with the BAPT20 and control groups (P < 0.05).In conclusion, BAPT improved the ram sperm quality via inhibiting the ROS production during cryopreservation, and could be applied as a promising supplement for ram semen cryopreservation.

Cryopreservation of margay (Leopardus wiedii) spermatozoa: Effects of different extenders and frozen protocols

Sperm cryopreservation offers many benefits to wild felids conservation programs. However, the implementation of these programs is limited by the different responses of each species to the cryopreservation protocols and extenders used, requiring the formulation of species-specific protocols. For this purpose, semen samples from 6 margays (Leopardus wiedii) were submitted to 2 cryopreservation protocols: 1) manual freezing (cooling rate of – 0.33 °C/min at 5 °C/180 min and freezing rate with two steps – 9 °C/min for 2 min and −19.1 °C/min for 2 min) and 2) automatic freezing machine (cooling rate of – 0.25 °C/min at 5 °C/120 min and freezing rate with one step −20 °C/min for 8.3 min) using 2 commercial extenders, an egg yolk-based (Test Yolk Buffer; TYB) and an egg yolk-free extender (AndroMed; MED). Post-thawed sperm quality was assessed at 3 time points (immediately after thawing and 1 and 2 h post-thawed) by sperm motility index (SMI), plasma membrane and acrosomal integrity, and mitochondrial membrane potential (MMP). Regarding SMI, TYB yielded superior results (29.4 ± 3.5%) compared to MED (11.2 ± 2.8%; p < 0.002) immediately after thawing until 2 h after thawing (TYB 3.9 ± 1.7% and MED 0.0 ± 0.0%; p < 0.05). Furthermore, the automated freezing method provided higher motility compared to the manual freezing procedure immediately post-thaw (25.08 ± 3.66% and 15.78 ± 3.29%, respectively) and 1 h post-thaw (13.71 ± 2.56% and 6.03 ± 1.97%, respectively; p < 0.05). The percentage of intact acrosomes and plasma membranes and the percentage of sperm with high MMP were superior for TYB when compared to MED regardless of cryopreservation protocol (p < 0.05). Conversely, the interaction between cryopreservation protocols and extenders was observed for MMP where TYB exhibits better results compared to MED (p < 0.05) in both procedures, but it was higher in automated procedures. For MED, no changes were found in MMP between procedures. Considering only TYB, samples showed higher MMP when submitted to an automated procedure (p < 0.05). In conclusion, the slow cooling rates with shorter time of exposure to glycerol contributed to minimize cryodamage in the Margays’ sperm. Moreover, results indicated that association between TYB and automatic freezing machine ensured the minimal quality of spermatozoa after thawing required for further use in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).

Effect of storage time on the quality of cauda epididymal spermatozoa of West African dwarf (WAD) rams

The objectives of the study were to develop a protocol for preserving Western African Dwarf (WAD) ram cauda epididymal semen that could be applied in preserving and storing the semen of endangered species/genetically valuable animals in case of death and to study the morphometric characteristic of epididymides after refrigerated storage. Thirty testes-epididymides were collected immediately after slaughter from mature WAD rams and transported in ice chest at (4.5–6 °C) to the laboratory. The samples were either processed immediately or stored in a refrigerator at 5 °C for 6, 12, 24, 48 h. The results indicate that the semen motility decreased (P < 0.05) when compared to the control [87.5 ± 2.1% (0 h), to 85.0 ± 1.8% (6 h), 73.3 ± 3.6% (12 h), 53.3 ± 2.5% (24 h) and 50.0 ± 2.9% (48 h)]. The sperm concentration also decreased (P < 0.05) as duration of storage increased (0 h) 157.5 ± 8.2, (6 h) 152.3 ± 5.8, (12 h) 125.3 ± 4.4, (24 h) 106.2 ± 2.9, (48 h) 98.5 ± 3.5. Semen viability decreased as duration of storage increased from the 0 h to 48 h [(P < 0.05; 84.0 ± 1.4%, 82.8 ± 2.2%, 77.3 ± 1.7%, 69.8 ± 1.5%, and 66.5 ± 1.2%, respectively]. Furthermore, there was a decrease (P < 0.05) in percentage of intact acrosome as duration of storage increased [(0 h) 90.7 ± 1.0%; (6 h) 89.3 ± 2.0%; (12 h) 85.5 ± 1.6%; (24 h) 70.0 ± 2.4%; and (48 h) 73.3 ± 2.1%]. The results from this study indicate that epididymal semen of WAD rams recovered and preserved at 5 °C for 48 h may be used for artificial insemination.

207 Cat oviductal exosomes as a tool to improve gamete rescue of endangered felids: effects on sperm capacitation and in vitro fertilization

Exosomes play important roles in reproduction, for example, facilitating fetal-maternal interaction and establishment of pregnancy. However, little is known about how oviductal exosomes affect sperm function. We investigated the effects of oviductal exosomes on sperm capacitation and fertilizing ability in domestic cats as a model for endangered felids. Oviducts were collected from cats (1-5 years) after elective spaying and flushed with 1mL of PBS. Exosomes (EX) were isolated using the Total Exosome Isolation kit (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) and labelled with BODIPY dye (boron dipyrromethene). Unattached dye was removed by using Exosome Spin Columns (MW 3000, Invitrogen). Presence and purity of EX was confirmed by transmission electron microscopy (TEM). To investigate the effects of EX on sperm capacitation, semen was recovered from epididymis of 5 cats (3 replicates) after elective neutering. One million spermatozoa mL−1 were incubated with or without EX (1V sperm to 2V total exosomes from 2 oviducts) for 1h in PBS. The samples were then incubated at 38.5°C in 1 of the 2 conditions: (1) capacitation (SOF medium+1000IU of penicillin, 10μg mL−1 streptomycin, 10μg mL−1 heparin, 20 µM penicillamine, 10 µM hypotaurine, 1 µM epinephrine); and (2) non-capacitation (capacitation medium without heparin, penicillamine, or hypotaurine) for up to 24h. Total motility, hyperactive and progressive motility, and percentage of intact acrosomes (FITC-PNA) were assessed at 0, 1, 2, 18, and 24h. Data were analysed by using SPSS software (IBM Corp., Armonk, NY) using a paired samples t-test with 95% CI. Vesicles of 30-100nm were observed by TEM, indicating successful isolation of EX, which were found to bind to both acrosome and mid-piece (BODIPY labelling). Sperm incubated with EX exhibited higher motility than those without EX (P&amp;lt;0.05 for all comparisons) at 1h (capacitation: 80±3.7v. 70±3.5; non-capacitation: 78±1.6v. 66±2.5%), 2h (capacitation: 77±1.3v. 61±1.1; non-capacitation: 74±0.9v. 55±3.5%), 18h (capacitation: 53±1.5v. 40±4.7; non-capacitation: 30±1.2v. 21±3.7%), and 24h (capacitation: 30±2.5v. 12±1.1; non-capacitation: 21±2.1v. 8±0.9%). Regarding progressive and hyperactive motility and acrosome integrity, only hyperactive motility at 1h using capacitation medium was significantly higher in the presence of EX than without it (18±1.5v. 9±2.7%; P&amp;lt;0.05). Next, we performed IVF using sperm with or without 1h incubation with EX. After 18h IVF, presumptive zygotes were stained with Hoechst 33342 and observed under a fluorescence microscope to assess fertilization and polyspermy. Preliminary data (30 oocytes/group) revealed that sperm incubation with EX reduced polyspermy (6±4% v. 20±8%) and improved normal fertilization (28±14vs 8±4%), although the differences were not significantly different (P&amp;gt;0.05 for both groups). In conclusion, the findings indicate that oviductal EX play roles in regulating sperm function by enhancing sperm motility. Further studies are needed to confirm the impact of EX on fertilization and how this strategy can be applied to endangered felid conservation.

Variation in post-thaw sperm quality of white-tailed deer bucks (Odocoileus virginianus) during rut

White-tailed deer farming is a growing industry in the United States, with breeding operations contributing significantly to the industry’s economic impact. Artificial insemination with frozen semen allows for selection and dissemination of valuable genetics, yet surprisingly little is known regarding the best time throughout rut to perform semen cryopreservation. The objective of this study was to compare semen quality following cryopreservation of white-tailed deer bucks collected early in the breeding season (September, n = 6), at peak rut (December, n = 8), and late season (March, n = 7). We hypothesized that post-thaw semen quality would be enhanced at peak rut. Mature bucks were anesthetized with tiletamine-zolazepam and xylazine administered intramuscularly via projector. Semen was collected by electroejaculation and cryopreserved using Optixcell extender. Overall and progressive sperm motilities were assessed for each sample before and after cryopreservation using a computer-aided sperm analyzer. Flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and DNA stability (acridine orange). Analysis of variance was applied to normalized data using a general linear mixed model with buck ID as a random variable, and a Tukey HSD test was performed as needed for post-hoc analysis. Pre-freeze overall and progressive sperm motilities were lowest in March, intermediate in September, and highest in December (p ≤ 0.04). Post-thaw overall and progressive motilities were lowest in September (p ≤ 0.02), but did not differ between December and March (p ≥ 0.12). The DNA Fragmentation Index was lowest in December, intermediate in September, and highest in March (p ≤ 0.05). The percentage of spermatozoa with intact plasma membrane was higher in December than September (p < 0.01), but the percentage of intact acrosomes per sperm with intact plasma membrane was highest in September (p = 0.03). This study confirms that post-thaw semen quality appears to be superior during peak rut (December) in bucks. Though semen collected early or late in rut may present acceptable motility, DNA stability is impaired, which could adversely affect fertility rates. This data suggests that semen cryopreservation during transitional periods should be avoided, though field studies evaluating the translation of these results into satisfactory pregnancy rates are warranted.

Use of alternative extender added of fructose aiming the cryopreservation of boar semen

This study aimed to analyze skimmed milk powder (SMP) and fructose in a new cooling curve to freeze boar semen. A total of 49 semen samples from seven boars were cryopreserved using the new curve with addition of glucose and fructose to the refrigerating diluents: Beltsville Thawing Solution (BTS + G; BTS + F) and Skimmed milk powder (SMP + G; SMP + F), totaling four experimental groups for analysis. To finish the curve, aliquots of semen were packaged in 0.5 ml straws and kept in liquid nitrogen. During the cooling curve, SMP mean spermatic vigor and motility were greater than the BTS (p &lt; 0.05). After thawing, a decrease of spermatic force and motility in both extenders was observed, where the BTS presented spermatic vigor (2.1 ± 0.55) and motility (38 ± 21.8), presenting better results (p &lt; 0.05). There was no statistical difference between sugars added to the BTS and SMP in spermatic force and motility (p &gt; 0.05), although the use of fructose allowed an equalization of motility between the SMP and BTS (p &gt; 0.05). Functionality of membrane was better preserved with the addition of fructose, in both extenders. The rate of sperm viability was significantly higher in extender containing glucose and SMP (71.8 ± 12.5). The percentage of intact acrosome was higher on the treatment containing glucose, independent of the extender (BTS + G: 81.8 ± 7.2, SMP + G: 81.4 ± 14.2). To conclude, the results suggest that the BTS is still the best option to cryopreserve and fructose could be used in boar semen cryopreservation in new cooling curve.

Effect of antioxidants supplementation on the quality of Beetal buck semen stored at 4°C.

Aim:An experiment was designed to evaluate the role of Vitamin E and glutathione in improving the seminal parameters during hypothermic storage of liquid semen at 4°C for 72 h.Materials and Methods:Thirty-six semen ejaculates were collected by artificial vagina from 6 bucks (Beetal) during the normal reproduction season (September to November) at weekly interval. The samples were centrifuged, and the seminal plasma was removed. The sperm pellet was diluted with Tris-based extender and divided into three groups. Group T1: Control samples without antioxidants, Group T2: Samples supplemented with tocopherol at 3 mM, and Group T3: Samples supplemented with glutathione at 1 mM. The samples were evaluated for progressive motility, percent liveability, percent abnormal spermatozoa, and acrosome integrity after liquid preservation for 0, 24, 48, and 72 h. The level of lipid peroxidation and antioxidant enzymes, namely, glutathione peroxidase (GPx) and superoxide dismutase (SOD) were estimated after liquid preservation for 0 and 72 h.Results:It was observed that, after storage of semen at 4°C up to 72 h, the progressive sperm motility, percent liveability, percent abnormal spermatozoa, and percent intact acrosomes were significantly (p<0.05) higher in group T2 and T3 samples as compared to control. However, the level of lipid peroxidation in T2 and T3 groups was significantly (p<0.05) lower after 72 h of incubation at 4°C. Similarly, GPx and SOD values were significantly (p<0.05) increased in T2 and T3 groups after 72 h of storage at 4°C.Conclusion:Thus, it can be concluded that Vitamin E and glutathione supplementation at 3 mM and 1 mM, respectively, while preserving the semen samples at 4°C helped in maintaining the seminal parameters up to 72 h and protected the spermatozoa from oxidative damage.

Interaction of extender composition and freezing method for effective semen cryopreservation in the North American river otter (Lontra canadensis)

Semen cryopreservation and storage in genome resource banks (GRBs), in combination with artificial insemination (AI), could be invaluable for genetic management and conservation of endangered otter species. For any applied conservation benefit, effective methods for otter sperm processing and cryopreservation first must be established. In this study, our objective was to develop an effective semen cryopreservation method for the North American river otter, evaluating the effect of extender composition (i.e., glycerol concentration, Equex STM paste supplementation) and freezing protocol (timing of glycerol addition, pre-freeze cooling rate, freezing/packaging method) on post-thaw sperm motility, longevity and acrosome status. Semen was collected from 14 otters housed at 9 zoos, and following cryopreservation in an egg-yolk based extender, thawed to assess sperm motility and acrosome status immediately post-thaw and during 6 h of in vitro culture. Results indicated that extender containing 4% glycerol was preferable (p < 0.05) to 8% glycerol but the temperature/timing of extender addition containing 4% glycerol did not affect (p > 0.05) post-thaw sperm parameters. Treatments with extender containing Equex and frozen by pelleting on dry ice showed greater (p < 0.05) motility and percentage of intact acrosomes compared to treatments frozen in extender without Equex, regardless of pre-freeze cooling rate. In the absence of Equex, pelleting provided superior post-thaw sperm motility (p < 0.01) and higher (p < 0.001) percentage of sperm with intact acrosomes compared to samples frozen in straws over liquid nitrogen vapor. Results of this study indicate that cryopreservation of otter sperm using an egg-yolk –TEST based extender containing 4% glycerol and 1% Equex, with the pellet freezing method, provided superior post-thaw sperm motility, longevity and acrosomal integrity compared to other combinations. Neither alterations in timing of glycerolated extender addition nor pre-freeze cooling rate had a discernable effect on post-thaw otter sperm parameters. These findings represent the first assessment of semen cryopreservation in any otter species and may be of value as a model for development of semen cryopreservation strategies in other endangered otter species.

Cryopreservation of canine sperm using egg yolk and soy bean based extenders

Animal protein-based extenders are widely used despite being a potential source of bacterial or mycoplasma contamination. Its replacement with vegetal protein-based extenders could represent an interesting alternative for dog sperm cryopreservation. This technique could be further improved by the addition of Tris-Glucose-Citric acid (TGC) that could physically protect the spermatozoa and improve its homeostasis. The aim of this study was to evaluate a cryopreservation protocol for dog spermatozoa using a soybean-based extender (LP1℗) as well as the effects of the addition of (TGC) immediately after the semen collection. Eleven ejaculates from purebred adult dogs were collected, centrifuged in the absence or presence of TGC and processed as fresh or cryopreserved spermatozoa with: egg yolk-based extender (CaniPRO) or LP1℗. Freezing the spermatozoa in LP1℗ reduced the amplitude of the lateral head displacement, the percentage of spermatozoa that showed the intact acrosome and the mitochondrial function (P<0.05). These samples also showed a trend towards increased percentage of apoptotic spermatozoa (P<0.05). The addition of TGC before centrifugation did not improve the seminal parameters and adversely affected motility (P<0.05) in the spermatozoa cryopreserved in CaniPRO. However, TGC did not affect motility and increased (P<0.05) the percentage of intact acrosomes in the spermatozoa cryopreserved in LP1℗, reaching similar values than those cryopreserved in CaniPRO.In conclusion, LP1® plus TGC provide the same level of protection to dog spermatozoa cryopreservation than the egg yolk based extender CaniPRO when comparing standard post-thaw sperm quality parameters.

191 SexedULTRA™, A NEW METHOD OF PROCESSING SEX SORTED BOVINE SPERM IMPROVES POST-THAW SPERM QUALITY AND IN VITRO FERTILITY

Since the first publications 30 years ago showing that flow cytometry was a reliable method to separate X and Y chromosome bearing sperm, the process has been subject to continual refinement. Numerous experiments have been performed in the last few years with the objective of developing an improved sex-sorted product, branded SexedULTRA™ (Sexing Technologies, Navasota, TX, USA) that retains sperm integrity to improve post-thaw sperm quality, in vitro embryo production, and field fertility compared with the previous XY method. Laboratory evaluations were performed on semen from 12 bulls at the Sexing Technologies laboratory in Navasota (TX, USA). Each ejaculate was divided in 2 aliquots and then processed in 1 of 2 methods (XY method or SexedULTRA™). Post-thaw sperm motilities were classified into percent total and progressively motile after thawing (0 h) and after a 3-h incubation at 37°C using a computer-assisted sperm motility analyzer (Hamilton Thorne IVOS II system, Hamilton Thorne Biosciences, Beverly, MA, USA). Percent intact acrosomes was also estimated after a 3-h incubation. Results were analysed by a mixed model ANOVA with the fixed effect of treatment and random effect of bull. Percent total motile SexedULTRA™ sperm was greater (P &lt; 0.001) than sperm processed following the XY method at 0 (78.8 v. 67.2%) and 3 h (51.0% v. 39.0%) post-thaw. Likewise, there was a higher percent of progressively motile sperm both at 0 (50.7 v. 44.9%) and 3 h (31.5 v. 4.4%) post-thaw in the SexedULTRA™ sperm. Percent intact acrosomes was also greater in SexedULTRA™ sperm compared with the sperm processed following previous method (78.0 v. 64.0%). In vitro fertilizations were performed as a measure of sperm competence using 8 ejaculates from 2 bulls in Sexing Technologies IVF laboratory in Laceyville (PA, USA). Five to 10 oocytes and 5,000 motile sperm/oocyte were placed per IVF drop for the analysis. A total of 3 straws and a minimum of 800 oocytes per treatment group (ejaculate × treatment) were included in the comparison for development to 8-cell stage (cleavage rate) and to Day 7 blastocyst stage, measured as total (grades 1 to 4) and freezable (grades 1 and 2) embryos. Results were analysed using a mixed model ANOVA with treatment as a fixed effect and bull, ejaculate within bull, and IVF cycle as random effects. Results from IVF trials are shown in Table 1. Total and freezable embryo numbers were significantly higher (P &lt; 0.05) when using SexedULTRA™ compared with XY sperm. Maintaining a suitable environment for sperm to progress through the various steps of the sex-sorting process results in better semen quality post-thaw as well as improved in vitro fertility. The SexedULTRA™ method confers a significant benefit in maintaining sperm integrity that, if translated into field fertility, could reduce the conception rate gap between conventional and sex-sorted bovine sperm. Table 1. Results from IVF and embryo culture using frozen-thawed, sex-sorted semen processed using the XY or the SexedULTRA™ method

Retained fertilizing capability in cryopreserved feline spermatozoa.

Sperm cryopreservation offers a long-term preservation of male genetic materials for future assisted reproductive technologies. However, dramatic changes in temperature during freezing and thawing injure sperm cells. While motility is essential for AI and membrane integrity is crucial for in vitro fertilization (IVF), sperm DNA integrity is a common index of fertilizing capability required for AI, IVF and intracytoplasmic sperm injection (ICSI). In endangered felids died unexpectedly, attempts have been made to recover as many DNA intact spermatozoa as possible from epididymis and testis to increase the opportunity to produce offspring in future. Although sperm from caudal epididymis has shown retained fertilizing capability after freezing and thawing (27.3% conception rate after unilateral intrauterine insemination), sperm recovery from the corpus epididymis has been suggested as an alternative to increase the amount of preserved genetic materials. To improve epididymal sperm quality, pre-treatment with single-layer centrifugation resulted in selection of sperm cells with intact DNA while post-thaw treatment with extracellular ATP incubation promoted the blastocyst rate. Cold storage of domestic cat testis for 7days at 4°C demonstrated <1% of sperm cells with fragmented DNA. Moreover, isolated testicular sperm cells, stored for 7days at 4°C, produced after ICSI poorer percentages of cleavage, morula and blastocyst than the fresh control. In wild felids, a death-to-necropsy time of 2hr after a jungle cat (Felis chaus) aged 10years died during anaesthesia plus another necropsy-to-sperm recovery time of 25hr has been reported to yield the post-thawed testicular sperm with 22.2% intact DNA. In summary, the chromatin structure of feline ejaculated and epididymal sperm seems to be tolerated to cold storage and cryopreservation; thus, fertilizing capability is well protected. In contrast, the cat testicular sperm DNA is generally damaged through the cryopreservation.

Mitigation of sperm tail abnormalities using demembranation approach in the clouded leopard (Neofelis nebulosa).

Clouded leopards (Neofelis nebulosa) produced high proportion of abnormal spermatozoa (mainly tail defects) that can limit sperm movement and conception. The study aimed to better identify the origin of those defects using a demembranation approach. Ejaculates (1-2 ejaculations/male; n=9) were allocated to simple washing (control; resulting in 11.7%±1.9% coiled tails) and processed through colloid centrifugation to reduce the number of sperm with tail defects (treatment, resulting in 5.9%±0.9% coiled tails). Aliquots of semen were incubated in hypo-osmotic solution (HOS, 60 mOsm fructose solution) containing 5mM dithiothreitol (DTT) (a reducing agent) to prevent oxidation of sperm membrane. Thereafter, 20% Triton X-100 (TX) (a detergent) was added to the HOS/DTT-treated samples. After HOS/DTT incubation, the control samples and sperm-selected samples presented 73.4%±3.1% and 73.9%±2.5% swollen sperm (bent and coiled) indicating membrane intact, respectively. Most of the coiled tail in the raw ejaculates could not be opened by TX indicating that the cause of coiled sperm tails may be from testicular origin. The proportion of sperm with tightly coiled tail tended to be lower in the sperm-selected group than control group (18.8%±3.8% and 26.5%±3.4%; p=.1), whereas the sperm opened up by TX tended to be higher in the sperm-selected group (53.6%±10.4% and 21.1%±7.9%; p=.06). The results indicated TX was able to uncoil half of the tightly coiled sperm in the semen undergone preparation. In conclusion, the coiled sperm in the clouded leopard semen were likely not a defect of sperm volume regulation during post-ejaculate (osmotic swelling) but pre-ejaculate origin. Semen preparation demonstrated its ability to lessen the primary sperm defects and selected spermatozoa that were prone to be mitigated after demembranation.

Natural honey as a cryoprotectant to improve Arab stallion post-thawing sperm parameters

ObjectiveTo investigate the effect of extender supplementation with different concentrations of a honey bee on post-thawed sperm motility, viability index, membrane and acrosome integrities in Arab stallion. MethodsFive ejaculates from each of four Arabian stallions were subjected to cryopreservation with a modified INRA-82, without any supplementation (control) or supplemented with 1%, 2%, 3%, 4% and 5% honey bee. After thawing, all samples were maintained at 37 °C, while analyses were performed at 0, 1, and 2 and 3 h. Sperm motility percentage, viability index percentage, membrane integrity percentage and acrosome integrity percentage of each sample were determined by conventional laboratory methods. ResultsRelative to the control group, supplementation with honey (2%, 3% and 4% significantly improved (P < 0.01 at least) post-thaw sperm motility, viability index (P < 0.001 at least) and had a positive effect on membrane integrity and intact acrosome percentage (P < 0.001 at least) at 0, 1, 2 and 3 h post-thawing. For all semen parameters, the lower concentration of honey (1%) and higher concentration (5%) did not show significant differences (P > 0.05) compared with the control. ConclusionHoney bee supplementation in Arab stallion semen extenders provided a better protection of sperm parameters against cryopreservation injury, in comparison to the control groups.

Evaluation of semen quality brought from Kundasale as replacement of locally produced semen for artificial insemination in Jaffna Peninsula, Sri Lanka

Artificial Insemination (AI) with frozen semen has been proved as the most potent method for rapid genetic improvement in domestic animals. While the use of AI with fresh semen is limited by the restricted period of sperm viability, this limitation may be overcome by the storage of frozen semen. Recently AI center of Thirunelvely is being advised to use the deep-frozen semen processed from Kundasale of Central Province (CP) for AI purposes. Thus the present study was undertaken to compare the quality of locally produced stored semen (LPS) and semen brought from Kundasale in deep-frozen condition (KDFS) by assessing the viability of them as the prominent factor that determine the successful of AI service . A batch of LPS and KDFS was obtained from the AI center at Thirunelvely. LPS was diluted with 2.9% sodium citrate buffer solution to evaluate the individual progressive motility and sperm velocity, sperm morphology, and acrosome integrity. Sperm smear was prepared by using 1% eosin and 0.75% Giemsa stain to identify intact and reacted acrosome and sperm abnormalities respectively. Thawed semen was also assessed for motility examination, percentage of intact acrosome, viability and morphology. Paired‘t’ test was performed to compare the means of the viability of LPS and KDFS (P Ving. Journal of Science 2012, Vol. 11, No. 1, 19-25

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos.

Gamete co-incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co-incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18h. In Experiment 1, only 1mmol melatonin showed lesser blastocyst rates than control (C: 23.2±6.7% versus 1mmol: 2.0±1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10nmol melatonin was significantly greater than controls (P<0.05). IVF media with 1mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.

Copy number variation of PRAMEY across breeds and its association with male fertility in Holstein sires

Multi-copy gene families are especially prevalent in the male-specific region (MSY) of the mammalian Y chromosome. Copy number variations (CNV) of these Y-linked gene families have been shown to affect human and animal fertility. The PRAMEY (Preferentially expressed antigen in melanoma, Y-linked) gene family is a newly identified, bovid-specific Y-linked gene family, which codes for a cancer/testis antigen that is expressed predominantly in testis and various tumors. The PRAMEY gene family is believed to play an important role in spermatogenesis and male fertility in cattle. The objective of this study was to investigate the CNV of PRAMEY within and across breeds and to determine whether CNV was associated with reproductive traits in Holstein bulls. A quantitative real-time PCR method was applied to measure the copy number of PRAMEY among 460 bulls using a Y-linked single copy gene, DDX3Y (DEAD box polypeptide 3, Y-linked), as a reference. The median copy number of PRAMEY was 13, ranging from 2 to 31. Significant variations in PRAMEY copy number were observed among 15 breeds investigated. Holstein bulls had the lowest median copy number (12), whereas Limousin bulls possessed the highest median copy number (26). Furthermore, bulls in the taurine lineage (13) had a significantly lower median copy number than those bulls in the indicine lineage (20). Association analysis revealed that PRAMEY copy number was correlated negatively with scrotal circumference (SC), relative scrotal circumference (RLSC), percentage of normal sperm (PNS), and nonreturn rate (NRR), but had no significant association with postthaw motility (PTM), incubated motility (IM), percentage of intact acrosome (PIA), sire conception rate (SCR), or relative breeding efficiency (RBE). The data from this study indicate that CNV of the PRAMEY gene family is associated with male reproductive traits and may serve as a valuable marker for sire fertility selection at an early age in cattle.

Methodological factors affecting the results of staining frozen–thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status

In the present study, some methodological factors affecting the acrosomal staining of frozen–thawed Japanese Black bull spermatozoa were investigated by examining; the effect of fixation/permeabilization procedure on intact acrosome percentage after fluorescein isothiocyanate peanut agglutinin (FITC-PNA) staining, the acrosomal staining patterns by using two types of fluorescent probes FITC-PSA (Pisum Sativum Agglutinin) and FITC-PNA and the effect of staining methods, either smear or vial, on intact acrosome percentage. Then intact acrosome percentage was compared between the samples stained by thus established method and those simply fixed with glutaraldehyde (glutaraldehyde fixation method). A possibility that FITC-PNA staining or the glutaraldehyde fixation methods could detect any difference in intact acrosome percentage or acrosomal staining patterns between fertile and subfertile bulls was also examined. The results showed that (1) 4% paraformaldehyde fixation plus 1% Triton X-100 permeabilization was better than absolute ethanol alone, (2) FITC-PNA acrosomal labeling was more specific than FITC-PSA, (3) sperm suspensions should be smeared and gently processed before acrosomal staining rather than spotted onto glass slides after staining in vial in order to avoid excessive mechanical damage of the sperm acrosome, and (4) staining spermatozoa with FITC-PNA had no major advantages over examination of simply glutaraldehyde fixed sperm samples and both failed to detect any significant difference in intact acrosome percentage between the fertile and the subfertile bulls used here. The present study demonstrates important methodological considerations which need to be taken into account in order to design a reliable and reproducible protocol for the study of the acrosome.