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This study aimed at scrutinizing efficiency of incorporating L-carnitine or M. oleifera leaves extract into semen diluent on improving cryopreservation capacity and in vitro fertilization ability of buck spermatozoa. Ejaculates (n =48) were collected by an artificial vagina from six adult Damascus bucks twice weekly during the breeding season (September–October). Following initial evaluation, ejaculates of each collection session from the same bucks were pooled, diluted (1:10) with glycerolized (3 % glycerol, v/v) tris-citric acid egg yolk diluent and were split into three aliquots. The first aliquot served as control, whereas the second and third aliquots were supplemented with 4 μL/mL L-carnitine and 400 μL/mL moringa leaves extract (v/v), respectively. Thereafter, all specimens were processed for cryopreservation and were stored in liquid nitrogen (-196 °C) for 12 months before post-thaw sperm criteria were analyzed by a computer-assisted sperm analysis (CASA) system. Integrity of sperm DNA post thawing was visualized in all semen groups by fluorescence imaging, and in vitro fertilization ability of spermatozoa was also determined. Inclusion of L-carnitine or moringa leaves extract into the diluent improved (P <0.05) post-thaw sperm physical, morphofunctional and kinematic attributes, whilst maintaining (P <0.05) integrity of sperm DNA throughout the freezing and thawing cycle. Consequently, both supplemented groups yielded higher (P <0.05) in vitro fertilization rates compared to control. These results accentuate the protective roles of these antioxidants on buck sperm against consequences of cryopreservation-induced oxidative stress, hence ameliorating post-thaw sperm quality and fertilization competence. This is crucial for successful application of AI and IVF in goat selective breeding programs. • Cryopreservation of buck spermatozoa is, to date, still challenging • This, in turn, impedes application of AI and IVF in goat breeding programs • Inclusion of L-carnitine or Moringa leaves extract in media enhanced sperm traits • It also efficiently maintained sperm DNA integrity post thawing • Both antioxidants yielded higher in vitro fertilization rates compared to control [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effects of replacing egg yolk extender with sesame oil on the quality of sperm in goats following incubation at 37°C, chilling at 4°C, and freezing. Semen samples were collected from four intact male goats. The individual semen sample was divided into six groups consisting of a control group and five treatment groups with different egg yolk-to-sesame oil ratios. Seminal plasma was removed, and the sperm pellet was diluted with experimental semen extenders. The control group contained an extender of 10 % egg yolk (SO0), and the experimental extenders were composed of 8.75 % egg yolk and 1.25 % sesame oil (SO1.25); 7.5 % egg yolk and 2.5 % sesame oil (SO2.5); 5 % egg yolk and 5 % sesame oil (SO5); 2.5 % egg yolk and 7.5 % sesame oil (SO7.5); and 10 % sesame oil (SO10). Each group of semen was divided into three groups, incubated at 37°C for 1 h, chilled at 4°C for 4 h, or frozen for 24 h. Five replicates were performed. Sperm quality was evaluated, including motility, viability, and functional membrane integrity. The SO1.25 group achieved the highest sperm quality rate among the treatment groups, and the extender did not have a negative effect compared to the control. However, the total replacement of egg yolk with sesame oil in an extender resulted in the lowest sperm quality. In conclusion, the ratios of egg yolk and sesame oil that were acceptable for goat semen cryopreservation were 8.75 % and 1.25 %, respectively. • The sperm quality did not differ between the 1.25 % sesame oil and control groups. • The 2.5 % sesame oil initially revealed an adverse effect on goat sperm. • The extenders in groups 5 %, 7.5 %, and 10 % sesame oil separated into two layers. [ABSTRACT FROM AUTHOR]

To assist in the conservation of collared peccary, it is important to strengthen semen processing protocols. The aim of this study was to compare the effects of different commercial extenders (BTS; NUTRIXcell+ and PRIMXcell Ultra) and TRIS + egg yolk on the functional and morphological aspects of collared peccary semen stored at 17 °C for 48 hours. Ten ejaculates obtained by electroejaculation were divided into 4 aliquots and diluted in the respective extenders, then stored in a biological incubator at 17 °C for 12, 24, 36, and 48 hours. The samples were evaluated for kinetic parameters, membrane functionality, membrane integrity, mitochondrial activity, morphology, and sperm-binding capacity. At the end of storage (48 h), promising results were found for motility parameters, with TRIS + egg yolk (71.0 ± 4.6%) being more efficient than NUTRIXcell+ (38.9 ± 10.9%) (P < 0.05) and similar to BTS (42.9 ± 11.9%) and PRIMXcell Ultra (46.8 ± 10.8%). The results for membrane integrity and mitochondrial activity were around ∼30–50%, with TRIS being the only extender to preserve both parameters (58.9 ± 5.3 and 59.2 ± 5.6%) for up to 48 hours, respectively (P < 0.05). Finally, the extenders could guarantee 60% membrane functionality and ∼ 60–70% normal sperm morphology, as well as similar binding capacity among the groups. In conclusion, TRIS + egg yolk is effective in preserving the sperm parameters of collared peccary semen at 17 °C for 48 hours, while PRIMXcell Ultra and BTS are viable alternatives for this purpose. • Cooling collared peccary semen at 17 °C is a promising approach for this species. • TRIS + egg yolk proved effective in preserving the functional parameters of cooling semen for up to 48 hours. • PRIMXcell Ultra and BTS are viable alternatives for cooling collared peccary semen. [ABSTRACT FROM AUTHOR]

Boar sperm is highly susceptible to cold damage. When temperature drops to 5°C, the plasmatic membrane is destabilized. The freezing process causes a reduction of the fertility window because frozen/thawed boar sperm has less survivability. The aim of this work was to analyze the effect on sperm characteristics and response to capacitation stimuli of cooling to 5°C using a controlled protocol. Also, we evaluated if the addition of Glycerol 2% or 3% at 5°C was able to modify these parameters. For this purpose, we assessed motility, plasmatic membrane integrity and acrosomal membrane status. Capacitation was induced using Tyrode´s capacitating medium (TCM) and assessed by chlortetracycline stain and induction of acrosomal reaction with Progesterone. Motility patterns were analyzed using a CASA system. These tests were performed at three different points of the freezing curve: 37°C; 17°C and 5°C. Response to TCM vs TBM was only significant at 37°C. While at 37°C and 17°C capacitated sperm was below 20%, at 5°C reached 50% both in the TBM and TCM. CASA analysis showed that spermatozoa exposed to TCM had higher LIN and WOB than those in TBM. All parameters were similar in the Glycerol concentrations studied. These results suggest that the chilling process may be causing an effect similar to cryocapacitation along the cooling curve, starting subtle at 17°C and reaching 50% of the sperm population at 5°C, being independent of Glycerol concentration. • Cryocapacitation of boar sperm starts at 17°C and affects 50% of sperm at 5°C. • Sperm exposed to TCM after cooling to 5°C show high linearity and low oscillation. • Glycerol didn´t modify response to capacitation and induced acrosomal reaction. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effect of the addition of different percentages of seminal plasma (SP) during the cooling at 5 °C of alpaca spermatozoa from vas deferens. Fifteen pools of sperm from vas deferens were evaluated and then divided into four aliquots that were diluted to a final concentration of 30 × 106 sperm/ml with either: (1) Tris with 20% egg yolk (T-EY) (control, 0% SP), (2) T-EY with 10% SP, (3) T-EY with 25% SP, and (4) T-EY with 50% SP. Samples were cooled at 5 °C and the following sperm parameters were evaluated after 24 and 48 h of storage: motility, viability, membrane function, acrosome integrity, morphology, and chromatin condensation. Motility was also evaluated after 72 h of storage. A significant decrease in progressive and total sperm motility was observed in samples cooled with 50% SP with respect to all diluted samples, while these parameters were preserved in samples cooled with 0%, 10%, and 25% SP. The percentages of sperm viability, normal morphology, and highly condensed chromatin did not change after the cooling process and were similar between cooled samples. Although a significant decrease was observed in the percentage of spermatozoa with functional membranes and with an intact acrosome in all refrigerated samples compared to raw sperm, the greatest decrease was observed in samples cooled with 50% SP. No advantage was observed from the addition of SP to alpaca spermatozoa obtained from vas deferens and being cooled. In addition, to preserve the sperm motility of cooled samples for up to 72 h, it should be recommended to include a 10% SP in the extender. • Study evaluates the cryoprotective effect of SP on alpaca sperm from vas deferens. • Seminal plasma addition before cooling did not change alpaca sperm motility pattern. • Samples cooled with 50% seminal plasma showed lower percentages of motile sperm. • Sperm viability did not change after cooling and was similar between cooled samples. • Sperm membrane function and acrosome integrity decreased in all cooled samples. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate the effects of crocin on canine sperm quality parameters during prolonged storage at 4 °C. Ejaculates from 10 dogs were diluted in a TRIS- egg yolk extender supplemented with 0 (control group), 0.5, 1, and 2 mM crocin and stored at 4 °C. Sperm membrane functional integrity, motility, and kinetics were assessed after 3 h, 24 h, 4 days and 7 days of storage. Based on the results, the more efficient concentration of crocin (0.5 mM) was chosen to evaluate sperm intracellular ROS levels, lipid peroxidation, and DNA fragmentation vs. the control. Semen with the addition of 0.5 mM crocin with respect to the control exhibited: i) increased (P < 0.05) sperm membrane functionality at 4 and 7 days of storage; ii) higher (P < 0.05) average path (VAP), straight-line velocities (VSL), and beat cross frequency (BCF) at 4 d of storage at 4 °C; iii) decreased (P < 0.05) intracellular ROS levels after 3 and 24 h storage. No differences in lipid peroxidation and DNA fragmentation were recorded between the control and C0.5 groups at any time point. Lipid peroxidation did not increase over time, while DNA fragmentation increased (P < 0.05) in both groups after 4 days of storage. The results demonstrated that the enrichment of extender with crocin improves to a certain extent canine semen quality, particularly after 4 days of storage at 4 °C. • Crocin supplementation preserves canine sperm viability and kinetics at 4 °C. • Crocin decreases sperm intracellular ROS levels during short-term storage. • Crocin delays the occurrence of sperm DNA fragmentation during prolonged storage. [ABSTRACT FROM AUTHOR]

Plant-based semen extenders, typically derived from soybean lecithin, are easier to modulate more and consistent in their composition than animal-based extenders. As large lecithin particles can, however, reduce effectiveness and solubility in bull semen extenders, sonication was used to create nano-lecithin (NL) particles of soybean lecithin. The objective was to determine the effects of lecithin type and concentration on the quality of frozen-thawed bovine sperm. We hypothesized that reducing the size of lecithin improves its interactions with the sperm and enhances the parameters that favor its motility, viability and fertility. Semen was collected from six mature Holstein bulls and ejaculates meeting minimum standards were pooled. Eight Tris-based extenders that contained 1, 2, 3, or 4 % of either conventional lecithin (L1-L4) or NL (NL1-NL4), plus two control extenders (one animal-based extender containing 20 % egg yolk [EY] and a commercial lecithin-based extender [BioXcell®]) were compared. Among soybean lecithin-based extenders, NL3 had the highest total and progressive sperm motility, and average path, straight-line and curvilinear sperm velocity, and was comparable to EY. Additionally, sperm mitochondrial activity was the highest in NL3, whereas sperm viability was highest in EY, NL3, and L4. Following in vitro fertilization of in vitro-matured bovine oocyes, NL3 had cleavage and hatching rates comparable to BioXcell®, but a lower blastocyst rate than EY. Overall, NL3 performed better than the other extenders for most end points, with efficiency comparable to EY. We, therefore, concluded that reducing lecithin particle size to a nano level improves sperm cryopreservation with optimal performance with 3 % NL. [ABSTRACT FROM AUTHOR]

Abstract The aim of this study was to assess the effect of different factors affecting vitrification success of donkey sperm: extender, sperm concentration, volume and storage vessel type. In Experiment 1, sucrose supplementations at 0.25 and 0.1 M were compared using two base extenders (containing or not egg-yolk); in Experiment 2, three sperm concentrations were assessed: 100, 200 or 300 million sperm/mL; and in Experiment 3, three different sperm volumes (100, 160 and 200 μL) and two different storage vessels (0.25 and 0.5 mL straws) were assessed. Sperm motility variables (CASA), plasma membrane and acrosome (evaluated under fluorescence microscopy) and sperm DNA integrity (flow cytometry) were evaluated after warming with comparisons of protocols. There was a greater total (55.7 ± 16.4%) and progressive (44.0 ± 11.5%) motility using the extender with egg-yolk and 0.1 M sucrose. There were no effects of sperm concentrations on vitrification results (P > 0.05). The 0.25 mL covered straw showed higher values than the 0.5 mL straw for total (50.0 ± 17.3% vs 2.0 ± 6.7%) and progressive (40.5 ± 14.9% vs 0.9 ± 1.5%) motility, plasma membrane (43.9 ± 14.4% vs 14.0 ± 16.4%) and acrosome integrity (51.5 ± 13.6% vs 28.0 ± 14.7%), respectively. In conclusion, values for donkey sperm quality variables after vitrification were greater using an extender containing egg-yolk and 0.1 M sucrose, at 300 million sperm/mL in 0.25 mL straws with outer covers. [ABSTRACT FROM AUTHOR]

Abstract The aim of the present study was to analyze the effect of dialysis on the quality of frozen/thawed buck semen. Ejaculates (n = 15) from three Saanen bucks were divided into three experimental groups. Semen in Group Ce (centrifugation) was processed by standard method and washed two times at 1.085 × g for 20 min. During this time, the diluted control semen (Co) was stored at room temperature. Semen in Group D was dialyzed using 300 kDa cut-off semi-permeable cellulose tubing. The semen from all groups was diluted with extender containing 20% egg yolk and frozen in liquid nitrogen vapor. After thawing, semen samples were evaluated by microscopic and biochemical analyses. Phospholipase A2 was in amounts that was 72.0 ± 11.7% less after dialysis and 21.3 ± 10.0% less after washing with centrifugation compared to the control semen (P < 0.05). Spermatozoa from Group Co had a lesser motility and viability and greater percentage of morphological abnormal spermatozoa in comparison to Groups D and Ce at 3 h after thawing and incubation on 37 °C. At the same time motility and percentage of HOST positive spermatozoa were greater in Group Ce compared with D (P < 0.05). There, however, was no difference in morphology and viability (CFDA/PI analysis) of spermatozoa between Ce and D group. Results from the present study suggest the dialysis is the promising alternative method for reducing phospholipase A2 in the buck semen before cryopreservation. [ABSTRACT FROM AUTHOR]

Diluents using components of plant origin have been developed as an alternative to animal based extenders for the dilution of bull semen, however, it is unclear if use of these diluents results in in vivo fertility rates similar to those that occur with use of traditional egg yolk-based diluents. The aim of this study was to assess the effect of semen diluent on 60-day non-return rate (NRR) following artificial insemination (AI) with frozen-thawed bull semen. The effect of semen dilution in one of three different commercial diluents (BullXcell – egg yolk-based, OptiXcell – plant-based or AndroMed – plant-based) on post-thaw total and progressive motility as well as kinematic parameters (Experiment 1) and field fertility (Experiment 2, n  = 1,480 inseminations) was assessed. Semen stored in OptiXcell had greater post-thaw total and progressive motility than AndroMed ( P  < 0.05) but did not differ from BullXcell. Semen stored in BullXcell had a greater beat cross frequency and straight line velocity compared to semen stored in AndroMed ( P  < 0.05) but did not differ when compared with use of OptiXcell; while values for these variables when using OptiXcell and AndroMed did not differ from each other ( P  > 0.05). There was no difference in any other sperm kinematic parameters ( P  > 0.05). There was no effect of diluent on 60-day NRR (71.5%, 67.8% and 70.6% for BullXcell, OptiXcell and AndroMed, respectively). In conclusion, while diluent significantly affected post-thaw sperm motility and kinematics, no effect on 60-day NRR was observed. Given that OptiXcell and AndroMed are animal protein-free media these diluents may be a suitable alternative to BullXcell for the storage of frozen-thawed bull semen. [ABSTRACT FROM AUTHOR]

As a non-threatened hystricognath rodent species, Spix ’s yellow-toothed cavies can be used as a model for the development of assisted reproductive techniques for the conservation of closely related species. The objective was to establish a functional protocol for cryopreservation of epididymal sperm from these cavies. Twelve sexually mature males, ∼2 y old and weighing ∼300 g, were euthanized. Sperm were recovered by retrograde flushing of the vas deferens and cauda epididymis with Tris extender. Thereafter, sperm were extended in Tris plus 20% egg yolk, with 3%, 6% or 9% glycerol or dimethyl sulfoxide (DMSO), placed in 0.25 mL straws and cryopreserved in liquid nitrogen. Sperm concentration, motility (using computer-assisted sperm analysis; CASA), plasma membrane integrity, osmotic response, morphology and sperm binding-ability were determined in fresh and frozen-thawed sperm. For most sperm endpoints, glycerol was a more desirable cryoprotectant than DMSO. Data (mean ± SEM) were similar with use of 3%, 6%, and 9% glycerol ( P  > 0.05) in osmotic response (40.66 ± 6.3%, 42.5 ± 7.1%, and 39.5 ± 5.0% respectably), and membrane integrity (55.17 ± 5.5%, 68.4 ± 4.1%, and 59.1 ± 4.9% respectably). Among concentrations assessed, the use of 6% glycerol resulted in the greatest ( P  < 0.05) post-thaw values for total motility (60.9 ± 4.4%), rapid subpopulation motility (27.7 ± 3.1%) and sperm-binding capability (227.0 ± 20.2). In conclusion, epididymal sperm from the Spix’s yellow-toothed cavies ( G. spixii ) are optimally cryopreserved in Tris extender with 6% glycerol and 20% egg yolk. [ABSTRACT FROM AUTHOR]

Anandamide (AEA), an endocannabinoid, has been shown to reduce capacitation and acrosomal exocytosis in human spermatozoa. Because buffalo spermatozoa are highly susceptible to cryopreservation induced damage, AEA was assessed as to whether it could protect spermatozoa from cryo-damage. Six ejaculates from six Murrah buffalo bulls (total 36 ejaculates) were utilized for the study. Each ejaculate was divided into four aliquots; spermatozoa in Aliquot 1 were extended in Tris-Citrate-Egg Yolk and frozen as per the standard protocol. Spermatozoa in Aliquots 2, 3 and 4 were incubated with AEA at 1 nM, 1 μM and 10 μM, respectively in Tris-Citrate extender for 15 min at 37 °C before cryopreservation. Cryopreserved spermatozoa were thawed at 37 °C for 30 s before assessment of sperm motility, membrane integrity, capacitation, acrosome reaction, mitochondrial membrane potential (MMP) and lipid peroxidation status. The proportion of motile and membrane intact spermatozoa were greater ( P <  0.05) with use of 1 μM AEA incorporated group compared with other groups. The proportion of un-capacitated and acrosome intact spermatozoa was greater ( P <  0.05) with use of 1 or 10 μM of AEA compared with the other groups. When compared to the control group, use of 1 μM AEA resulted in a greater proportion of spermatozoa with high MMP ( P <  0.05). There was no significant difference in the lipid peroxidation status of spermatozoa among any of the four groups. It was inferred that the protective role of AEA during cryopreservation of buffalo spermatozoa was dose dependent and incubation of spermatozoa with AEA at 1 μM concentration prior to cryopreservation reduced cryo-capacitation and improved post-thaw sperm quality in buffalo. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare sperm DNA fragmentation of frozen-thawed epididymal sperm of dogs using the SCSA (Sperm Chromatin Structure Assay) and SCDt (Sperm Chromatin Dispersion test). For this purpose, epididymis from neutered dogs were minced and incubated in a Tris-based extender. The recovered sperm were frozen in a two-step cooling protocol with Tris-based, egg yolk extender and increasing glycerol concentrations, and stored in liquid nitrogen. After thawing, each replica was incubated at 38 °C for 24 h. Sperm DNA fragmentation index (sDFi) was assessed by SCSA and SCDt at 0, 3, 6 and 24 h of incubation and compared within treatments. The relationship and agreement between techniques were evaluated by Pearson’s coefficient and Intraclass Correlation Coefficient (ICC). The results were expressed as mean ± standard error of the mean (SEM). Both techniques indicated there was a significant increase of DNA fragmentation after 24 h of incubation. Moderate correlation (r = 0.65; P <0.01) but lack of agreement (ICC = 0.451; P >0.05) was found between SCSA and SCDt. The lack of agreement indicates that SCSA and SCDt measure different aspects of DNA fragmentation. Four halo morphologies were detected after 24 h of incubation using the SCDt: un-fragmented DNA with a small halo, fragmented DNA with large halo and two new halo presentations never described before for dog sperm: receding sperm with a disappearing halo and “bald” sperm without chromatin dispersion halo around the core. Sperm without a halo of chromatin dispersion are not described by the manufacturer and are similar to un-fragmented sperm, which could lead to erroneous results when using the SCDt. Further studies with different incubation periods and including the new morphologies described in this study should be performed. In conclusion, although SCSA and SCDt can evaluate the changes in the sperm DNA fragmentation dynamics of frozen-thawed epididymal dog sperm, these provided different findings on sperm DNA fragmentation. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate the effect of the association between glycine-milk (GM) based extenders made with different concentrations of soy lecithin (SL) and freezing rates (FR) on semen quality after thawing. Pooled semen from rams (n = 12) were diluted in GM extenders with 20% egg yolk (EY–20%) or with different concentrations of SL: 0.5% (SL–0.5%), 1.0% (SL–1.0%), and 2.0% (SL–2.0%). The diluted semen (150 ×106 spermatozoa/0.25 mL) was frozen at three FR of − 10, − 20, and − 60 °C/min, and subsequently thawed and analyzed. Results revealed that EY–20% and SL–2.0% had better kinetic parameters, and showed higher proportions of viable, non-apoptotic, plasma-membrane-intact spermatozoa (A-/PI-) and non-capacitated spermatozoa (F), and had lower acrosome-reacted spermatozoa (AR) in the EY–20% and satisfactory values for SL-2.0% compared to SL–0.5% and SL–1.0% (P < 0.05). The FR at − 20 and − 60 °C/min maintained higher A-/PI- and viable spermatozoa compared to − 10 °C/min. The combination EY–20% and − 60 °C/min showed the highest A-/PI- and F (P < 0.05) and the lowest AR, and it did not differ from the combinations EY–20% at − 20 °C/min and SL–2.0% at − 20 °C/min (P > 0.05). In conclusion, the combination EY–20% and − 60 °C/min, showed the best cryoprotective effects on ram spermatozoa. Changes in spermatozoa after thawing were related to the use of the type of extender, the amounts of the same compound in the extender, and the freezing rates to which they were subjected. • Use of extenders and freezing rates are related to spermatozoa quality after thawing. • Higher extender osmolality was related to better cryoprotection in ram spermatozoa. • Extender with 2.0% soy lecithin can be used in ram spermatozoa cryopreservation. • Fast freezing rates have a better cryoprotective effect on ram spermatozoa. • Extender with 20% egg yolk and fast freezing rate preserves spermatozoa structure. [ABSTRACT FROM AUTHOR]

In the study, melatonin, a known antioxidant pineal peptide was used as an additive in the tris-egg yolk glycerol-based semen extender in Hariana bull semen and post-thaw sperm characters were evaluated. In the study, Group I was a control without melatonin; and Group II, III, and IV were having 0.5 mM, 1 mM, and 2 mM melatonin/80 × 106 spermatozoa, respectively were treatment groups. Thirty-two semen ejaculates from 4 Hariana bulls were processed for freezing and post-thaw sperm characteristics were evaluated. Sperm motility, velocity, the viability with intact membrane, and total antioxidant capacity were markedly (P < 0.05) improved in Group IV compared to all other groups. The lipid peroxidation and total protein carbonylation were substantially (P < 0.05) decreased in Group IV compared to all other groups. The population of cryocapacitated, acrosome-reacted, and apoptotic-like spermatozoa were significantly (P < 0.05) decreased in Group IV. Further, the relative band intensity of 74 kDa protein and percent of spermatozoa showing positive immune reactivity to tyrosine-phosphorylated proteins was decreased in Group IV. The progesterone-receptor ligand binding, in vitro capacitation response, and Vanguard distance were markedly (P < 0.05) improved in Group IV. In summary- Group IV having 2 mM melatonin was found to be optimal in providing cryoprotective effects to Hariana bull spermatozoa after freezing-thawing and can be suitably used as a semen additive during semen cryopreservation. • Melatonin in semen extender lowers oxidative damage to spermatozoa. • Melatonin in semen extender lowers cryocapacitation-like changes in spermatozoa. • Melatonin ion semen extender minimizes apoptotic-like changes in spermatozoa. • 2 mM melatonin can be used as an additive in Tris-egg yolk based semen extender. [ABSTRACT FROM AUTHOR]

The present study was conducted to investigate a suitable source (Expt. 1) and concentration (Expt. 2) of plasma egg yolk (PEY) and concentration of camel skim milk (CSM; Expt. 3) to supplement tris based extender for chilled storage of dromedary camel semen. In Expt. 1, PEY (20%) of six avian species (domestic chicken, domestic duck, Japanese quail, partridge, pigeon and guinea fowl) was added to semen extender. In Expt. 2, different concentrations (0, 10, 20, 30 and 40%) of selected PEY from Expt.1 were added to semen extender. In both Expt. 1 and 2, CSM remained constant (20%). In Expt. 3, semen extender was supplemented with different concentrations of CSM (0, 20, 40, 60 and 80%) while the concentration of PEY remained constant. The sperm viability parameters were assessed at 6, 12 and 24 h following chilled storage. In Expt. 1, progressive forward motility (PFM) of diluted semen supplemented with pigeon PEY was similar to domestic duck and Japanese quail PEYs (P > 0.05) and superior to other PEYs (P < 0.05). In Expt. 2, PFM following the addition of 20% pigeon PEY was similar to 10 and 30% (P > 0.05) and greater than 0 and 40% (P < 0.05). In Expt. 3, total motility, PFM and live percentage of sperm were better in 20% compared to 40, 60 and 80% CSM (P < 0.05). In the last experiment, PFM in 20% was better than 0% CSM (P < 0.05). In conclusion, pigeon PEY at the concentration of 20% and CSM at the concentration of 20% could provide beneficial effect on some of the sperm viability parameters during chilled storage of dromedary camel semen. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effects of 8% virgin coconut oil (VCO) combined with different percentages of egg yolk in Tris extender on the quality of chilled and frozen-thawed bull semen. A total of 24 ejaculates from four bulls were collected using an electroejaculator. Semen samples were diluted with 8% VCO in Tris extender which contained different concentrations 0% (control), 4%, 8%, 12%, 16% and 20% egg yolk. The diluted semen samples were divided into two fractions: one was chilled and stored at 4 °C until evaluation after 24, 72, and 144 h; the second fraction was processed by chilling for 3 h at 4 °C to equilibrate, then packaged in 0.25 ml straws and frozen and stored in liquid nitrogen at −196 °C until evaluation after 7 and 14 days. Both chilled and frozen semen samples were then thawed at 37 °C and assessed for general motility using computer-assisted semen analysis (CASA), viability, acrosome integrity, and morphology (eosin–nigrosin), membrane integrity (hypo-osmotic swelling test) and lipid peroxidation (thiobarbituric acid-reactive substances (TBARS)). The results indicate treatments with 8%, 12%, 16% and 20% egg yolk with 8% VCO had greater sperm quality ( P < 0.05) as compared with the control. The treatment with 20% egg yolk had the greatest sperm quality ( P < 0.05) among the treated groups for both chilled and frozen-thawed semen. In conclusion, the use of 8% VCO combined with 20% egg yolk in a Tris-based extender enhanced the values for chilled and frozen-thawed quality variables of bull sperm. [ABSTRACT FROM AUTHOR]

Cholesterol-loaded cyclodextrin (CLC) is known to improve ram sperm cryosurvival. This study expands on previous research to: (1) determine the mechanism by which CLC improves ram sperm cryosurvival and (2) compare the efficiency of a novel, skim milk-based extender containing CLC to a traditional egg yolk-based extender. Hypothesis #1 was that CLC enhances membrane cholesterol content to increase the resistance of ram sperm to cold and osmotic stress, thereby improving cryosurvival. We first assessed the ability of fresh sperm treated with CLC to withstand cold shock. Second, fresh sperm were treated with CLC to evaluate their tolerance to osmotic stress. Third, to confirm that cholesterol is incorporated into the sperm using CLC, we quantified sperm cholesterol. To test Hypothesis #2 that CLC is most effective in a medium without competing cholesterol, we compared sperm cryosurvival and fertility in skim milk-based extender containing CLC versus in a traditional egg yolk-based freezing extender without CLC. Our data confirmed that CLC treatment improves ram sperm cold shock and osmotic stress resistance, and augments sperm cholesterol content. Semen in skim milk-based extender containing CLC prior to freezing, had more motile sperm with intact acrosomes after thawing compared to semen in egg yolk-based extender. In contrast, sperm plasma membrane integrity and in vivo fertility of the semen cryopreserved in the skim milk-based extender with CLC did not differ from semen that was cryopreserved in egg yolk-based extender. Further research is warranted to combine CLC with other cryoprotection strategies or to modify the insemination protocol. [ABSTRACT FROM AUTHOR]

The current study determined the effect of the egg-yolk (phospholipid source) level (egg yolk [20% EY] vs. skim-milk + egg yolk [SM + 2% EY]), cryoprotectant (glycerol [Gly] vs. glycerol + methylformamide [Gly + MF]), and pre-freeze cooling rate (–0.1 vs. –1 vs. −5 °C/min) on post-thaw stallion sperm quality. In Experiment 1, ejaculates (n = 27) from 9 stallions (3 ejaculates each) with varied sperm quality (High, Average, or Low) were frozen in EY-Gly, SMEY-Gly, EY-Gly + MF, or SMEY-Gly + MF extenders. Sperm in each group were cooled from 22° to 5°C using either −0.1 °C/min or −1 °C/min linear cooling rates prior to freezing. In Experiment 2, ejaculates (n = 24) from 12 stallions (2 ejaculates each) with High or Average sperm quality were frozen in EY-Gly, EY-Gly + MF, or in BotuCrio (BC) extenders. Sperm in each group were cooled from 22° to 5°C using either –1 or −5 °C/min linear cooling rates prior to freezing. In Experiment 1, for stallions with High or Average sperm quality, either cooling rate generally resulted in lower sperm quality for the SMEY-based extenders than for the EY-based extenders (P < 0.05). Stallions with Low sperm quality were unaffected by any experimental treatment (P > 0.05). In Experiment 2, a −5 °C/min cooling rate yielded lower sperm quality in BC than in EY-Gly or EY-Gly + MF groups (P < 0.05); however, a −1 °C/min cooling rate yielded similar sperm quality among these treatments (P > 0.05). In summary, the phospholipid level in the freezing extender and the pre-freeze cooling rate, but not the penetrating cryoprotectant, affected the post-thaw quality of stallion sperm. • 20% egg yolk (EY) yielded higher post-thaw stallion sperm quality than 2% EY. • Glycerol or glycerol and methylformamide yielded similar post-thaw sperm quality. • Pre-freeze cooling rate influenced post-thaw sperm quality in 2% EY-extenders. • Pre-freeze cooling rate did not affect post-thaw sperm quality in 20% EY-extenders. • Pre-freeze cooling rate was not affected by penetrating cryoprotectants. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effect of two commercial extenders, AndroMed® (AM) and Androstar® Plus (AS) both with and without the addition of egg-yolk (EY), for cooling llama semen. A total of sixteen ejaculates were collected from four males. Each ejaculate was divided into four aliquots and diluted with: AM, AM with 20 % EY (AM-EY), AS and AS with 20 % EY (AS-EY) and then cooled to 5 °C in an Equitainer®. Evaluations were carried out in raw semen, after dilution (0 h) and after 24 and 48 h of cooling. Data were analysed using either Friedman or ANOVA. Although total motility decreased in all cooled samples compared to the corresponding 0 h (P < 0.05), the highest percentages were observed in AM-EY being significantly higher than all other cooled samples after 24 h and higher than AS and AS-EY after 48 h. No significant differences were observed in the percentages of live acrosome-intact sperm between extenders at all times tested. A significant decrease in the percentage of sperm membrane osmotic function was observed in samples cooled with AS and AS-EY after 24 and 48 h vs. raw semen and in AM 48 h vs. raw semen. Finally, a significant increase in the percentage of sperm with abnormal tails was observed in the samples cooled with AS and AS-EY. Of all the extenders used, AndroMed® could be considered an option for cooling llama semen and the addition of EY to this extender improves its effectiveness. The data that support the findings of this study are available from the corresponding author upon reasonable request. • Post-cooling sperm viability was similar between extenders. • Sperm membrane function decreased and tail abnormalities increased with Androstar®. • The addition of egg yolk to AndroMed® improved its efficacy for cooling llama sperm. [ABSTRACT FROM AUTHOR]

Flow cytometry can be used to evaluate many sperm attributes and Dr. Duane Garner was influential in developing assays to understand sperm physiology and function. We review some of Dr. Garner's work and describe experiments that evaluate sperm capacitation using Dr. Garner's philosophy. In exploratory experiments, boar sperm were cryopreserved in lactose egg yolk (LEY) or Beltsville Freezing Extender 5 (BF5) and incubated in one capacitating medium. In another experiment, frozen-thawed bull sperm were incubated in TALP-Ca or CFDM1 capacitating media. In both experiments, sperm viability and capacitation were evaluated using multiple probes. Boar sperm frozen in LEY had greater survival rates (38%) than sperm frozen in BF5 (22%; P < 0.05) but did not capacitate as effectively as sperm in BF5 (P < 0.05). In Experiment 2, bull sperm survived to a greater extent when incubated in TALP-Ca than in CFDM1 (P < 0.05) and had greater capacitation for most parameters (P < 0.05). Of particular interest, 77% of sperm incubated in TALP-Ca had activated second messenger systems involved in capacitation, compared with < 5% of sperm incubated in CFDM1. The results indicate different freezing and capacitating media induce different responses to sperm capacitation and functions. If only sperm viability and acrosomal integrity were evaluated, these results would be interpreted very differently. Dr. Garner's philosophy of evaluating multiple sperm parameters was an impetus to determine unique treatment differences which help in understanding sperm capacitation, and design further experiments to determine how media content causes sperm physiology differences. • Boar cryopreservation medium will affect the ability of sperm to capacitate. • Capacitation medium modulates the response of bull sperm to capacitate in vitro. • The characteristics of sperm capacitation can be evaluated flow cytometrically. [ABSTRACT FROM AUTHOR]

Since the inception of bovine semen cryopreservation, egg yolk and milk based extenders have been used to protect sperm from the detrimental effects of cooling and freezing. In recent years, demand for alternatives to conventional commercial extenders has arisen as the risk of introducing exotic diseases through transporting egg yolk based products has been recognized. Egg yolk can also interfere with sperm evaluation and the presence of particulate material in the extender may reduce fertility. Soybeans contain lecithin, a phospholipid fraction that can substitute for high molecular weight lipoprotein and phospholipids from egg yolk and prevent or ameliorate damage to the sperm plasma membrane that occurs during extension, cooling, and cryopreservation. Soy lecithin based extenders have been evaluated for processing and freezing bovine semen, although extender from soybean milk has not been studied as extensively. Commercially available soy lecithin based extenders are used increasingly but remain under scrutiny and are not universally accepted. With these observations in mind, this review is intended to examine effects of conventional cryopreservation procedures, methods of assessment, and potential for developing soybean extract as an acceptable alternative to traditional egg yolk and milk based extenders for bull sperm cryopreservation. [ABSTRACT FROM AUTHOR]

The objectives of the present study were to compare the use of soybean-based (Andromed), liposome-based (Optixcell), and egg yolk-based (Ovine Red, Triladyl, and Biladyl) extenders for cryopreservation of white-tailed deer semen. In experiment 1, ejaculates obtained from six bucks were aliquoted into the following extenders: Andromed, Ovine Red, Triladyl, and Biladyl (containing 4%, 6%, or 8% of glycerol). In experiment 2, ejaculates obtained from eight bucks were divided amongst Andromed, Ovine Red, and Optixcell extenders. Total and progressive sperm motility were assessed for each sample before and after cryopreservation using a computer-automated semen analyzer. In experiment 2, flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and chromatin stability (acridine orange). In experiment 1, both Andromed and Ovine Red extenders exhibited higher post-thaw total motility than Biladyl containing 4% or 6% of glycerol ( p < 0.05). Andromed also produced higher progressive motility than all other extenders ( p < 0.01) before and after cryopreservation with no differences amongst the other extenders ( p ≥ 0.11). In experiment 2, there were no differences in total and progressive motility between Andromed, Ovine Red, or Optixcell extenders ( p ≥ 0.39). Additionally, there were no differences in sperm viability ( p = 0.18), acrosome integrity in viable sperm ( p ≥ 0.10), or DNA fragmentation index ( p = 0.15). These results demonstrated that soybean (Andromed) and liposome-based (Optixcell) extenders are equally as effective at cryopreserving white-tailed semen as egg yolk-based Ovine Red extender, but are superior to egg yolk-based Biladyl or Triladyl extenders. [ABSTRACT FROM AUTHOR]

The aim of the current study was to evaluate the effects of different concentrations of rainbow trout seminal plasma (RTSP) (0.1%, 1% and 10%) in extenders containing either egg yolk or lecithin for use in Awassi ram semen cryopreservation. Pooled sperm were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in egg yolk or lecithin extender containing no RTSP, 0.1%, 1% or 10% RTSP (v/v). Semen samples were assessed for sperm motility, plasma membrane integrity [hypoosmotic swelling test (HOST) and Hoechst 33258] and defective acrosomes [FITC-conjugated Pisum sativum agglutinin (PSA-FITC)] at the following five time points: after dilution with extender A; after equilibration; and post-thaw at 0 h, 3 h and 5 h. Malondialdehyde (MDA) was examined only after thawing. Freezing and thawing procedures (dilution, equilibration and post-thaw incubation at 0 h, 3 h and 5 h) negatively affected the motility (P < 0.001) and acrosome integrity (P < 0.001). Additionally, freezing and thawing negatively affected the plasma membrane integrity, as determined by the HOST and Hoechst 33258 (P < 0.001). The extender group affected the motility (P < 0.001) and the HOST results (P < 0.001). Levels of MDA in the eggy olk extender with 1% RTSP group were significantly lower than in the lecithin control group (P < 0.05). In conclusion, the egg yolk extender groups that were supplemented with 10% and 1% RTSP provided greater cryoprotective effects for semen survivability during 5 h incubation than the other extender groups. [ABSTRACT FROM AUTHOR]

The aims of this study were to evaluate the effects of anti-oxidant butylated hydroxytoluene (BHT), when added at different concentrations into lecithin-based Bioxcell ® (BX) and two egg-yolk-based; Tris (TY) and citrate (CE) semen extenders, on post-thaw bull sperm quality and oxidative stress. A total of 30 ejaculates from three bulls were collected using an electro ejaculator. Ejaculates were extended with one of the BX, TY and CE extenders, which contained different concentrations (0.0 – control, 0.5, 1.0, 1.5, 2.0 and 3.0 mM/ml) of BHT. The extended semen samples were chilled to 4 °C, and then frozen slowly to −196 °C in 0.25 ml straws before being stored in liquid nitrogen for 2 weeks. Results showed that supplementation of BHT improved ( P < 0.05) general motility, progressive motility, morphology, acrosome integrity, DNA integrity and malondialdehyde of sperm at 0.5 mM/ml for BX and at 1–1.5 mM/ml of BHT for TY and CE when compared with the control. However, greater concentrations of 2.0 and 3.0 mM/ml of BHT had a detrimental ( P < 0.05) effect compared with the control with all extenders evaluated. In conclusion, BHT supplementation at lesser concentrations (0.5–1.5 mM/ml) could improve frozen–thawed bull sperm quality by reducing oxidative stress produced during the freezing–thawing procedures in either lecithin or egg-yolk based extenders. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effects of two different egg yolk extenders incubated with or without Sperm Talp on the motility and plasma membrane integrity of cryopreserved bovine epididymal spermatozoa after freezing. Twenty-five testicles with epididymides from mature bulls were collected at the abattoir. Epididymal sperm recovery was performed by retrograde flushing using a skim milk-extender (Botu-Semen™). After recovery, sperm were incubated either without or with Sperm Talp and then submitted to centrifugation. For the freezing process, half of the testes were processed with Tris egg yolk extender, and half were processed with Botu-Bov™ egg yolk extender. Samples incubated in Sperm Talp exhibited better results than epididymal spermatozoa that were incubated without Sperm Talp ( p < 0.05). Both Botu-Bov™ and Tris could be utilised to freeze sperm from the bovine epididymides if the sperm were previously incubated with Sperm Talp. The extenders examined in this work did not differ in their effect on plasma membrane integrity after freezing. [ABSTRACT FROM AUTHOR]

Demand for alternative of egg yolk in freezing extenders have increased in recent years due to variability in egg yolk composition, risk of microbial contamination and presence of steroid hormones. The alternative to egg yolk-based extender (EY) can be soya lecithin-based extender (SL) and liposome-based extender (LP). However, the efficacy of SL is still a matter of debate. Few studies have been performed on the effect of LP but to date evaluation of buffalo semen cryopreserved in LP has not been studied. Therefore, this study was designed to compare SL and LP with conventional EY for evaluation of post-thaw quality of buffalo semen. Results showed that total, progressive and rapid sperm motility were found significantly higher ( P < 0.05) in LP among these extenders. In vitro assessment of post-thaw sperm longevity has also resulted in better maintenance of sperm kinetics and motility in LP in comparison to other extenders. Furthermore, sperm cryopreserved in LP travelled significantly more ( P < 0.05) distance in cervical mucus as compared to SL and EY. Therefore, it can be concluded that the LP is more efficient than SL and EY for the cryopreservation of buffalo semen. [ABSTRACT FROM AUTHOR]

This study was designed to identify the most effective non-permeable cryoprotectant (CPA) for the cryopreservation of rabbit semen by comparing the effects of different concentrations of low-density lipoproteins (LDL) on post-thaw sperm quality with those of whole egg yolk or sucrose. In a second experiment, the performance of the non-permeable CPAs identified as most effective was assessed in vivo by determining reproductive performances. Pooled semen samples were diluted to a ratio of 1:1 (v:v) in freezing extender (Tris-citrate-glucose and 16% dimethylsulfoxide as permeable CPA) containing as non-permeable CPAs 6, 8, 10 or 15% LDL from egg yolk, 0.1 M sucrose, or 15% egg yolk. The semen was loaded in 0.25 mL straws and frozen in liquid nitrogen vapor. After thawing, we determined sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Our results clearly revealed a significant effect of LDL concentration on semen quality. Also, at an optimal concentration of 10%, motility and acrosome integrity were improved over the values recorded for egg yolk ( P < 0.05). Based on the in vitro data, 3 groups of does ( n = 30 each) were inseminated with fresh semen or semen frozen using sucrose or 10% LDL. Sucrose led to a significantly higher conception rate than LDL and reproductive performance was similar to that observed for fresh semen. Our findings indicate the markedly better performance of sucrose in vivo as a non-permeable CPA for the cryopreservation of rabbit semen. [ABSTRACT FROM AUTHOR]

The aim of the present study was to evaluate the effect of the holding time at 15°C prior to cryopreservation (2, 4 and 8h), thawing rate (37°C for 20s or 70°C for 8s) and post-thaw incubation temperature (15°C or 37°C) on the post-thaw boar sperm quality. These are important time periods in the freezing–thawing process which have been less studied. Sperm-rich ejaculate fractions from three healthy boars were collected once a week for five consecutive weeks and were cryopreserved with the lactose-egg yolk extender (LEY). Sperm quality was determined by assessing the motility, the acrosome status, and the sperm plasma membrane integrity at 30, 150 and 240min of incubation. The results show that with the holding time at 15°C prior to cryopreservation there was not a clear effect until at least 24h of holding time. The thawing rate and the post-thaw incubation temperature, however, had a marked effect on sperm quality. When the samples were thawed at 70°C for 8s, the sperm viability, motility and some kinetic variables (VCL, VSL, VAP and ALH) were greater than with results observed when the samples were thawed at 37°C for 20s. In addition after thawing the sperm samples incubated at 15°C had a sustained sperm quality for longer, up to 4h post-thawing. [ABSTRACT FROM AUTHOR]

Abstract: Reactive oxygen species generated during the freeze–thawing process may reduce sperm quality. This study evaluates the effects of melatonin supplementation as an antioxidant in the semen extender on post-thaw parameters of bull spermatozoa. Melatonin was added to the citrate–egg yolk extender to yield six different final concentrations: 0, 0.1, 1, 2, 3 and 4mM. Ejaculates were collected from six proven Holstein bulls. Semen was diluted in the extender packaged in straws, which was frozen with liquid nitrogen. The semen extender supplemented with various doses of melatonin increased (p <0.05) total motility, progressive motility, linearity, sperm track straightness, lateral head displacement, viability, integrity of the sperm membrane and total normal morphology of sperm after the freeze–thawing process. The most effective concentration of melatonin in microscopic evaluations of the bull sperm freezing extender was 2mM. The highest (p <0.05) value of total antioxidant capacity (48.9±2.7) and the lowest value of lipid peroxidation (2.7±0.8) were achieved by inclusion of 3mM concentration of melatonin in the semen extender and the highest activity of catalase (0.7±0.1) was obtained by 2mM melatonin. Four millimolar concentration of melatonin were reduced (p <0.05) the progressive motility and straight linear velocity. In conclusion, supplementation of 2 or 3mM concentration of melatonin in the semen extender improved the quality of post-thawed semen, which may associate with a reduction in lipid peroxidation as well as an increase in the total antioxidant capacity and antioxidant enzyme activity. [Copyright &y& Elsevier]

Abstract: The aim of this study was to assess biologically safer components as alternatives to egg yolk for the frozen storage of ram semen using casein, coconut or palm oil in either Salamon's diluent (S) or a swim-up medium (SU). Ejaculates were frozen as pellets and sperm motility (subjectively) and acrosome integrity (FITC-PNA/PI) by flow cytometry were assessed at 0, 3 and 6h after thawing and incubation at 37°C. Three experiments were done: different concentrations of palm oil (5%, 10% and 20%); casein added as emulsifier and protective agent; and differences between egg yolk, coconut and palm oil in S and SU. 20% of oil added to SU accounted for a lesser percentage (P <0.05) of motile cells compared to rest while no differences were found between different oil levels on viable cells. When casein was added to diluents containing 5% of palm oil, no differences were found between palm or casein (P >0.05). No differences were found when S and SU were compared neither as groups nor between S alone and containing coconut or palm oil; however, SU alone yielded less motility than SU 5% coconut. However, in both groups, S and SU, egg yolk accounted for the greatest values in both bases. These results indicate that none of biologically safer media components (casein, palm or coconut oil) used in this study maintained the function of ram spermatozoa after freeze-thawing better than S-containing egg yolk. The application of vegetable oils as substitutes for egg yolk in diluents for the cryopreservation of ram spermatozoa requires further research. [Copyright &y& Elsevier]

Abstract: Several studies reveal that vitamin E acts as a cellular stabilizer of unsaturated lipids against oxidative deterioration, thus maintaining structural and functional integrity at the subcellular level. The objective of this study was to evaluate Vitamin E (Trolox) addition to freezing extender for ram spermatozoa. Semen samples were diluted in Tris-yolk egg medium without antioxidant (control group) and with Trolox in different concentrations (30, 60 and 120μM). After thawing (37°C/30s), samples were subjected to analysis for plasma membrane integrity (PMi), acrosome integrity (Aci), mitochondrial membrane potential (MMP), sperm kinematics, and ultrastructural integrity. The Trolox 60 and 120μM groups showed higher percentages of iPMs (P <0.05) when compared to the control group. Differences were observed among groups in sperm kinematic indicators (progressive motility, linearity, straightness, oscillation index, straight-line velocity and average path velocity), with higher values (P <0.05) for the Trolox 60 and 120μM groups. On ultrastructural assessment, Trolox addition at the three concentrations preserved spermatozoon head plasma membranes, while for the spermatozoon tail, plasma membrane preservation at 60μM was higher (P <0.05) than the other groups. The Trolox 60 and 120μM groups presented more mitochondrial ultrastructural preservation than the other groups (P <0.05). These results indicate that Trolox addition to Tris-egg yolk at 60 and 120μM provides greater structural integrity (plasma membrane and mitochondria) and kinematics for ram spermatozoa after cryopreservation. [Copyright &y& Elsevier]

Abstract: The objective of this study was to evaluate the effect of addition of egg yolk (EY) or soy lecithin (SL) based extenders on dog sperm parameters during 10days storage at 5°C. Four ejaculates of pooled semen from three Mongrel dogs were divided into three aliquots and extended to a final concentration of 200 million sperm/mL using following non-commercial extenders: egg yolk extender (EYE) group – semen+20% Tris-EYE; soy lecithin (SL) group 1 – semen+SL extender with 0.04% SL; SL group 2 – semen+SL extender 0.4% SL. The extended semen samples were stored at 5°C and were evaluated for sperm mitochondrial membrane potential (MMP), plasma membrane integrity (%PMI) and computer assisted sperm analyzer (CASA) sperm motility parameters on days 0, 1, 2, 3, 4, 7 and 10. The extender, days of storage and extender×days of storage affected the MMP, %PMI and CASA sperm motility parameters (P <0.05). The addition of 0.4% SL extender reduced the speed of deterioration of sperm parameters evaluated in this study compared to EY and 0.04% SL extenders (P <0.05). In conclusion, the addition of 0.4% SL extender enhanced sperm preservation based on the variables evaluated in the present study compared to EYE and 0.04% SL extenders and plausibly preserves sperm quality longer. [Copyright &y& Elsevier]

Abstract: This study was conducted to determine the effect of antioxidants on standard semen parameters, lipid peroxidation and fertility of Boer goat semen after cryopreservation. Ejaculates from four bucks were collected, evaluated and pooled at 37°C. The pooled semen was diluted with Tris citric acid fructose for washing. Semen samples, which were diluted with a Tris-based extender containing the antioxidant ascorbic acid (8.5mg/ml), butylated hydroxytoluene (2mM), cysteine (5mM) and hypotaurine (10mM) and an extender without antioxidant supplementation were cooled to 4°C and frozen in 0.25 straws with programmable freezer and finally stored in liquid nitrogen. Data (10 replicates) were analyzed by one-way analysis of variance. Mean (±SEM) progressive motility was significantly higher in ascorbic acid than other supplement groups and control samples (P >0.05). Best values were observed in ascorbic acid followed by BHT, cysteine, and hypotaurine. Antioxidant supplementation in extender showed significant (P <0.05) better values than the control group for sperm membrane integrity, acrosome integrity and viability. The ability of antioxidants to reduce the lipid peroxidation (LPO) after freeze thawing was measured by the formation of malondialdehyde (MDA) using the thiobarbituric acid method. Results showed that addition of antioxidants significantly reduced the rate of LPO in comparison to control (P <0.05). Ascorbic acid exhibited better values (1.27±0.28), than butylated hydroxytoluene, cysteine and hypotaurine 1.32±0.42, 2.27±0.16 and 2.38±0.17 respectively, which are significantly better than control (3.52±0.54). Higher pregnancy rate was observed with ascorbic acid followed by butylated hydroxtolune, hypotaurine and cysteine. However, differences in the fertility rate were non-significant with hypotaurine, cysteine and control groups. [Copyright &y& Elsevier]

Abstract: The use of chilled semen has raised increased interest due to several reasons. The present study compares 3 extenders for chilling of canine semen over a 10 days storage period. Sperm rich fractions (n =30) were divided into three aliquots and diluted with (a) a self-made tris-egg yolk extender (TEY), (b) the commercial CaniPRO™Chill 10 (CP, Minitüb, Germany) and (c) the Uppsala Equex-2 system (UpA+B): UpA+B (n =12) or UpA (n =18). % of totally and progressively motile sperm using computer-assisted sperm analysis (CASA, TM, PM), % live, membrane intact spermatozoa (SYBR-14/propidium iodide), % morphologically abnormal sperm, % acrosomal changes (Spermac®) and % membrane intact sperm (HOS test) were examined on days 0, 1, 2, 3, 5, 7 and 10. Semen quality significantly decreased during storage with significantly lower motility and lower % of live, membrane intact spermatozoa in UpA+B diluted samples. Although quality of samples chilled with TEY, CP and UpA was similar within the first 3 days, only TEY and CP revealed good results in preserving semen quality over 10 days with significantly higher PM, CASA motility parameters and % morphologically normal sperm in CP-chilled samples (each p <0.05). [Copyright &y& Elsevier]

Abstract: The aim of the present study was to evaluate the cryoprotectant effect of different non-permeating sugars for boar sperm. Pooled semen from three boars was used for the experiments. In the first experiment, the sperm quality of boar sperm cryopreserved with an egg-yolk based extender supplemented with different monosaccharides (glucose, galactose or fructose) was compared to a control cryopreserved in lactose–egg yolk extender. In the second experiment, the effect of five disaccharides (lactose, sucrose, lactulose, trehalose or melibiose) on boar sperm cryosurvival was studied. Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150min at 37°C after thawing: percentages of sperm with intact plasma membrane (SIPM), sperm presenting high plasma membrane fluidity (HPMF), sperm with intracellular reactive oxygen substances production (IROSP) and apoptotic sperm (AS). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. Freezing extenders supplemented with each of the monosaccharide presented smaller cryoprotective effect than the control extender supplemented with lactose (P <0.05). However, from the three monosaccharides tested, glucose provided the best sperm quality after freezing–thawing. With respect to the disaccharides studied, samples frozen with the extender supplemented with lactulose exhibited in general the lowest sperm quality, except for the percentage of capacitated sperm, which was highest (P <0.05) in the samples cryopreserved with the trehalose extender. Our results suggest that disaccharides have higher cryoprotective effect than monosaccharides, although the monosaccharide composition of the disaccharides is also important, since the best results were obtained with those disaccharides presenting glucose in their composition. [Copyright &y& Elsevier]

Abstract: The objective of this study was to identify possible specificity in the extender formulation for the cryopreservation of ram spermatozoa recovered from three origins (ejaculate, electroejaculate or epididymis), by evaluating post-thawing sperm quality and fertility. Ejaculated, electroejaculated or epididymal spermatozoa samples obtained from identical rams (8) were cryopreserved in four different extenders (TES-Tris–fructose with one of two egg yolk concentrations: 10% Y10 and 20% Y20, and with one of two glycerol rates: 4% G4 and 8% G8). Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability with SYBR-14/PI and acrosomal status with PNA/PI). Spermatozoa obtained by electroejaculation were of poorer quality after freezing/thawing, demonstrating that protocols for these samples need to be optimized. Egg yolk at 20% was more appropriate for freezing sperm from any of the sources. In general, 4% glycerol improved the quality of post-thawing samples recovered from ejaculate and electroejaculate, while 8% glycerol was more appropriate for samples recovered from the epididymis. Based on these results, an analysis of fertility was conducted. Fertility rates were similar between ewe groups inseminated with post-thawed sperm obtained from two sources: ejaculate (cryopreserved in Y20+G4), and cauda epididymis (Y20+G8), and this rate was less in the electroejaculated sample (Y20+G4). [Copyright &y& Elsevier]

Abstract: The aim was to assess the in vitro effect of glycerol, ethylene glycol or acetamide on frozen-thawed ram spermatozoa. Aliquots of each sixteen ejaculates from four rams of the Morada Nova breed were diluted in Tris-egg yolk with glycerol (5%), ethylene glycol (3% or 5%) or acetamide (3% or 5%) and frozen at −196°C. After thawing, progressive sperm motility was greater (P <0.05) in cryopreservation with glycerol 5% and ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Acrosome integrity was greater (P <0.05) with ethylene glycol 5% than acetamide (3% or 5%). The percentage of sperm without oxidative stress was greater (P <0.05) with ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Plasma membrane integrity was greater with glycerol 5% (P <0.05) than with the other cryoprotectants. Thus, it is concluded that glycerol 5% and ethylene glycol 3% or 5% protect ram sperm against the harmful effects of freezing and that glycerol 5% offers greater protection to sperm plasma membrane. [Copyright &y& Elsevier]

Abstract: The objective of this study was to design an AI protocol using cooled semen to obtain pregnancies in the llama. Each raw ejaculate was subdivided into four aliquots which were extended 1:1 with: (1) 11% lactose–egg yolk (L–EY), (2) Tris–citrate-fructose–egg yolk (T–F–EY), (3) PBS-llama serum (S-PBS) and (4) skim milk-glucose (K). Each sample reached 5°C in 2.5h and remained at that temperature during 24h. Percentages of the semen variables (motility, live spermatozoa) in ejaculates and samples cooled with L–EY were significantly greater than those obtained when cooling with the other extenders; therefore this extender was used (1:1) for all inseminations. Females were randomly divided into four groups (A–D) according to insemination protocol. Group A: females were inseminated with a fixed dose of 12×106 live spermatozoa kept at 37°C. Group B: females were inseminated with a fixed dose of 12×106 live spermatozoa, cooled to 5°C and kept for 24h. Group C: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C and kept for 24h. These groups (A–C) were inseminated between 22 and 24h after induction of ovulation. Group D: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C, kept for 24h and AI was carried out within 2h after ovulation. Pregnancy rates were 75%, 0%, 0% and 23% for groups A, B, C and D respectively. These results indicate that it is possible to obtain llama pregnancies with AI using cooled semen and that the success of the technique would depend on the proximity to ovulation. [Copyright &y& Elsevier]

Abstract: This study was carried out to investigate if the substitution of chicken egg yolk (CEY) with duck egg yolk (DEY) in extenders can improve the quality of frozen-thawed semen of Nili-Ravi buffalo bulls and to study if reducing DEY level in extender affects the freezability results. Thirty semen samples collected from three buffalo bulls were diluted in extenders A, B, C, D and E containing tris, citric acid, fructose, egg yolk, glycerol and antibiotics. Extender A contained 20% CEY (control), while extenders B, C, D and E contained 5, 10, 15 and 20% DEY, respectively. After freezing and storage for 24h in liquid nitrogen, samples were evaluated for post-thaw quality. The post extension sperm motility did not differ between extenders A (control) and E (20% DEY). The same was true for post-thaw percentage of sperm with functional plasma membrane and percentage of sperm with abnormal heads or mid pieces. However, extender E showed higher (P <0.05) values for post-thaw sperm motility, livability and absolute index of livability of spermatozoa at 37°C compared to extender A. Spermatozoa with abnormal tail were lower (P <0.05) in extender E compared to extender A. Values of these parameters of post-thaw semen quality were highest for extender E containing 20% DEY and decreased significantly with decrease in the concentration of DEY, except sperm abnormalities (head, mid-piece and tail) which increased with decrease in DEY level. These results showed that replacement of 20% CEY with 20% DEY in extenders significantly improved post-thaw sperm motility, livability and absolute index of livability of spermatozoa and reduced tail abnormalities. Reduction in the level of DEY in extenders from 20% adversely affected post-thaw semen quality of Nili-Ravi buffalo bulls. [Copyright &y& Elsevier]

Abstract: The objective of this study was to determine whether replacing the egg yolk with soybean lecithin in the Botu-Crio® cryodiluent would maintain the fertility of cryopreserved stallion sperm. Two experiments were performed to evaluate cell freezability. In experiment 1, sperm from 15 stallions were frozen in Botu-Crio® (BC) or Botu-Crio® which contained 45g/L soybean lecithin (BCLS45) in place of the egg yolk. In experiment 2, we compared different concentrations of soybean lecithin: 0, 10.0, 12.5, 15.0, 17.5 and 20.0g/L (BC, BCLS10, BCLS12.5, BCLS17.5 and BCLS20, respectively). In experiment 1, sperm frozen in BC and BCLS45 exhibited similar (P >0.05) percentages of total motile sperm (61% and 61%, respectively); progressively motile sperm (27% and 27%, respectively) and sperm with intact plasma membranes (IMP; 53% and 57%, respectively). Similarly, sperm frozen in BC or BC containing any concentration of soybean lecithin maintained similar (P >0.05) percentages of total motile sperm (61–68%) and progressively motile sperm (27–31%). In the first fertility trial, we used cryopreserved semen from a single stallion was inseminated into mares. The semen from the sperm that were frozen in BC diluent resulted in a higher fertility rate (66%, 16/24) compared to the sperm that were frozen in BCLS45 diluent (17%, 5/29; P <0.01). Similarly, in a second fertility trial, the mares that were inseminated with the sperm that were frozen in BC diluent exhibited a higher fertility rate (66%, 16/24) compared to the mares that were inseminated with the sperm that were frozen in BCLS20 (40%, 10/25; P <0.05). Finally, in a third trial, the sperm that were frozen in BC resulted in a higher fertility rate in mares (75%, 18/24) compared to the sperm that were frozen in BCLS10 (41%, 10/24; P <0.05). Although replacing the egg yolk in the BC cryodiluent with soybean lecithin provided similar laboratory results for stallion sperm, after cryopreservation, the sperm that was frozen with soybean lecithin in the diluent correlated with lower fertility rates. Based on these results, we concluded that the use of BCLS can be used as an alternative diluent for cryopreserving stallion sperm. However, the resulting reduced fertility rate is a matter of concern. Further studies are necessary to clarify the reasons for this decrease in fertility and to determine the optimal lecithin concentration for diluents to freeze stallion sperm. [Copyright &y& Elsevier]

Abstract: The aim of this study was to determine the effect of butylated hydroxytoluene (BHT), a lipid-soluble anti-oxidant added in different concentrations to the Tris egg yolk extenders on semen cytological parameters pre freezing and post thawing (motility, morphology, viability, acrosome integrity and membrane integrity) of Boer goat spermatozoa. A total of 40 ejaculates from four Boer goat bucks were collected using an artificial vagina. Ten replicates of the ejaculates were diluted with a Tris egg yolk based extender which contained various concentrations (0.5mM, 1.0mM, 2.0mM and 3.0mM) of butylated hydroxytoluene while one sample was processed without supplementation of antioxidant and served as control. The diluted semen was cooled at 4°C and loaded into the straw and then stored in liquid nitrogen. It was evident that supplementation of BHT produces positive effect in terms of motility, membrane integrity and acrosome integrity in comparison with the control group in cooled and frozen Boer goat semen. Results showed significant differences in motility, membrane integrity, acrosome integrity and viability of cooled and frozen Boer goat spermatozoa at different concentrations. Motility, membrane integrity, acrosome integrity and viability was significantly higher in all treated groups than the control group (P <0.05) while there was no significant differences (P >0.05) in morphology trait between all group in cooled semen. However, improvement (P <0.05) was observed only in terms of the membrane integrity and acrosome integrity compared to the control and other treated groups in frozen semen. In conclusion, BHT can be used in cryopreservation of Boer goat semen in order to reduce the oxidative stress on spermatozoa. [Copyright &y& Elsevier]

Abstract: The aim of this study was to evaluate the effects of cold storage and egg yolk concentration on the distribution of spermatozoa within the different subpopulations. Twenty ejaculates from 4 dogs were collected, diluted in either TRIS buffer containing 20% (TEY20) or 10% centrifuged egg yolk (TEY10) and cooled following a conventional protocol. The kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates and after 24 and 72h of preservation at 5°C. A multivariate clustering procedure separated 54,261 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (19.80%), Subpopulation 2 consisting of slow and low-linear spermatozoa (25.21%), Subpopulation 3 consisting of high speed and progressive spermatozoa (23.88%), and Subpopulation 4 consisting of highly active but non-progressive spermatozoa (31.11%). Although, cold storage had a significant (P <0.05) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after cold storage. Subpopulations 1 and 2 significantly (P <0.001) decreased during cold storage (Subpopulation 1: 26.6, 16.9 and 18.4%; and Subpopulation 2: 33.6, 21.3 and 24.0%, respectively, for fresh, 24 and 72h post-cooled), whereas Subpopulations 3 and 4 significantly (P <0.05) increased (Subpopulation 3: 16.7, 27.6 and 24.3%, and Subpopulation 4: 23.1, 34.1 and 33.4%, respectively, for fresh, 24 and 72h post-cooled). Regarding the relative percentage of spermatozoa within each extender, Subpopulation 3 was more frequently observed in TEY20 after both 24 and 72h of cold storage. Significant correlations (P <0.05) were found between the proportions of spermatozoa assigned to Subpopulation 3 in the fresh ejaculates and those in stored samples after 24h (r =0.48498). In conclusion, cold storage significantly modified both the specific parameters and the distribution of spermatozoa within subpopulations. These changes did not affect the general motile sperm structure present in dog, which is conserved during cold storage. The analysis of the changes observed in structures of subpopulations also suggests that the TEY20 provide more effective preservation of dog semen during cold storage. [Copyright &y& Elsevier]

Abstract: Chicken egg yolk is held as an excellent cryoprotective agent for freezing canine semen. Recent advances have enabled the extraction of low density lipoproteins from egg yolk, which are responsible for the cryoprotective abilities of the latter. The objective of this article was to compare 3 semen extenders for freezing canine semen: 2 containing egg yolk (Tris egg yolk and Equex STAMP) and one containing 6% LDL. After freezing and thawing 20 ejaculates from 5 different dogs, the 6% LDL extender produced 50% mobile spermatozoa, compared with 48% with the Equex® extender and 27.7% with the extender containing egg yolk alone (EY). In vitro functional tests demonstrated that the integrity of the plasma membrane (hypoosmotic test) was respected in 65–66% of spermatozoa as a function of the extender; DNA integrity was respected in more than 97% of the spermatozoa. The Equex® extender provided superior acrosome integrity (FITC/PSA test): 68.4% compared with 55.1% with LDL and 53.3% with egg yolk. However, the 6% LDL extender resulted in fewer spermatozoal anomalies (Spermac® test), with 54.6% normal spermatozoa compared to 53.6% for Equex® and 53.3% with the egg yolk. All six of the bitches inseminated artificially via the intra-uterine route (Scandinavian technique) using semen frozen in the 6% LDL extender became pregnant. The LDL extender resulted in percentages of mobile spermatozoa and movement characteristics that were as good if not better than those obtained with the reference extenders following thawing. The 6% LDL extender appears to have the same cryoprotective qualities as the reference diluent, Equex® STAMP. [Copyright &y& Elsevier]

Abstract: Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. Under the present study, the cryoprotective effect of taurine and trehalose on buffalo sperm quality parameters after freeze–thaw process was studied. Buffalo semen was cryopreserved in tris-based egg yolk extender along with cryoprotectants like taurine (50mM) or trehalose (100mM) and used for the assessment of sperm quality parameters like motility, viability, plasma membrane integrity, total antioxidant status and the extent of cryocapacitation. The results were compared to semen cryopreserved in tris-based egg yolk extender only as control. Post-thaw semen evaluation clearly indicated that the addition of taurine or trehalose significantly improved (P <0.05) the motility, viability and membrane integrity compared to control spermatozoa. The extent of sperm cells underwent cryocapacitation was significantly lowered (P <0.05) in presence of taurine or trehalose. Moreover, the percentage of in vitro capacitated cells in the treated samples was comparable to the control spermatozoa along with maintaining other sperm quality parameters. Finally, compared to the control and trehalose treated sample, addition of taurine to the freezing extender showed more positive effect on the total antioxidant power of seminal plasma and spermatozoa. It is concluded that the addition of taurine or trehalose to the freezing extender led to the reduction of cryodamage to the buffalo spermatozoa. [Copyright &y& Elsevier]

Abstract: The primary goal of this study was to compare the effects of 5% ethylene glycol (EG) and 5% glycerol (G) on fertility of frozen–thawed dog semen following intravaginal insemination. The sperm-rich fraction of the ejaculate of three male dogs was collected, pooled and divided into two aliquots, and then frozen with a Tris–glucose–egg yolk–citric acid extender containing either 5% G or 5% EG. A total of 10 bitches were inseminated twice, five with G-frozen–thawed semen and five with EG-frozen–thawed semen; intravaginal inseminations were performed the 4th and the 5th day after the estimated LH peak; four straws, thawed in a 37°C water bath for 1min and diluted in a Tris buffer, were used for insemination (200×106 spermatozoa); the insemination dose was introduced in the cranial vagina of the bitch using a sterile plastic catheter. Ovariohysterectomy was performed in all bitches between days 29 and 31 after the calculated LH surge, and pregnancy status, and the number of conceptuses and corpora lutea were recorded. All bitches were pregnant. Neither the number of conceptuses, nor the ratio of conceptuses to corpora lutea (conception rate) was significantly different between groups. In this first screening, with a limited number of bitches, EG-frozen semen did not show a higher fertility than G-frozen semen when used for two intravaginal inseminations. Irrespective of the semen used, conception rate was 0.50. [Copyright &y& Elsevier]