Effect of butylated hydroxytoluene on bull spermatozoa frozen in egg yolk-citrate extender

Effect of butylated hydroxytoluene (BHT) on the quality of frozen-thawed Holstein bull sperm in egg yolk-citrate extender was evaluated. High quality semen samples were diluted in egg yolk-citrate extenders containing 0, 0.5, 1, 2 and 4 mM BHT and subsequently frozen in liquid nitrogen. Pre-freeze and post-thaw progressive motility, and live/dead ratio and acrosomal integrity of 200 sperm per slide, stained with Eosin-Nigrosin and Giemsa, were evaluated at 0, 2 and 4 h after thawing. There was a significant decrease in forward motility, livability and acrosomal integrity up to 4 h after thawing the frozen sperm. Upon thawing, sperm progressive motility at 1 mM BHT was significantly (P<0.001) higher (11%) than other groups, but percentages of live sperm and live sperm with intact acrosomes were higher at 0.5 mM BHT. BHT at 4 mM BHT caused a significant decrease in motility, livability and acrosomal integrity during preparatory stages of freezing sperm. It is concluded that 0.5-1.0 mM BHT can be beneficial for freezing Holstein bull spermatozoa in egg yolk-citrate diluent, when inseminated immediately after thawing.     

Resveratrol supplementation and cryopreservation of buck semen

Resveratrol (Resv) was tested to assess its effects on buck semen freezability. Ejaculates of 4 bucks were collected, washed and diluted in a commercial extender at 30 °C. Extended semen was divided into 4 aliquots supplemented with increasing concentrations of Resv: 0 μM (control); 10 μM; 25 μM and 50 μM. Aliquots were cooled to 4 °C in 5h and frozen in LN₂. Thawing was performed at 37 °C for 30 s. At the 3 stages of the experiment (30 °C, 4 °C, thawing), motility (CASA), osmotic resistance (Hos test) and integrity of cytoplasm and acrosome membranes (PI/PSA staining) were assessed. Moreover, in thawed samples, the oxidative status (MDA assay) and early apoptosis (DNA fragmentation by TUNEL assay) were evaluated. Resveratrol supplementation did not affect most of the motility parameters analysed, except for total motility, ALH (lateral head displacement) and velocity distribution (P < 0.05). Functional and morphological integrity of membranes was not affected at any stage of the experiment (P > 0.05). In thawed spermatozoa, the oxidative status was not preserved by Resv (P > 0.05) while early apoptosis, was significantly decreased in the 50 μM Resv group (P < 0.05). Resveratrol did not improve buck semen freezability; the observed effects on motility and DNA were not dose dependent and not mediated by a potential anti-oxidant activity.     

The effect of seminal plasma on alpaca sperm function

In order to advance the development of assisted reproductive technologies in alpacas and other Camelids, the objective of this study was to explore the role of seminal plasma concentration on motility and functional integrity of alpaca sperm. Sixteen male alpacas > 3 y of age were used. In Experiment 1, epididymal sperm were incubated for 0 to 6 h in 0, 10, 25, 50, or 100% seminal plasma and motility was assessed. In Experiment 2, epididymal sperm were incubated in 0, 10, or 100% seminal plasma for 3 h and motility, acrosome integrity and DNA integrity were assessed. In Experiment 3, ejaculated sperm were incubated in 10, 25, 50, or 100% seminal plasma for 0 to 6 h and motility assessed. In Experiment 4, ejaculated sperm were incubated in 10 or 100% seminal plasma for 3 h and motility, acrosome integrity, DNA integrity, and viability were assessed. Epididymal and ejaculated sperm maintained motility longer when incubated in the presence of 10% seminal plasma compared to 0, 25, 50, or 100% seminal plasma (P < 0.001). The mean ± SEM percentage of epididymal sperm with intact acrosomes was less (P < 0.001) in samples incubated in 0% seminal plasma (39.4 ± 3.73) compared to 10% (75.3 ± 1.20) or 100% (77.4 ± 0.90) within 1 h after incubation. However, DNA integrity of ejaculated and epididymal sperm was not significantly affected by seminal plasma concentration. The mean viability of ejaculated sperm was reduced in the presence of 100 (12.7 ± 2.33) compared to 10% (36.2 ± 4.68) seminal plasma (P < 0.001) within 1 h of incubation. We concluded that alpaca semen should be diluted to a final concentration of 10% seminal plasma to prolong motility, preserve acrosome integrity, and maintain viability of sperm.     

Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30-60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 × 10⁶ sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 × 10⁶ sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 × 10⁶ sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.     

Effect of low density lipoproteins in extender on freezability and fertility of buffalo (Bubalus bubalis) bull semen

This study was designed to determine whether low-density lipoporoteins (LDLs) extracted from egg yolk in extender improve the freezability and fertility of buffalo bull semen. Semen from three Nili-Ravi buffalo bulls was diluted at 37 °C with tris-citric acid extender (50 × 10⁶ motile spermatozoa mL⁻¹) containing LDLs 2.5%, 5%, 10%, and 15% extracted from egg yolk and extender containing 20% egg yolk was kept as control. Diluted semen was cooled to 4 °C in 2 h, equilibrated at 4 °C for 4 h, filled in 0.5 mL French straws, and kept on liquid nitrogen vapors for 10 min. Straws were then plunged and stored in liquid nitrogen (-196 °C). Sperm motility (visually; %), plasma membrane integrity (%; with supravital hypo-osmotic swelling test), and viability (%; with dual staining test using Trypan-blue Giemsa) were assessed at post-dilution, pre-freezing and post-thawing. At post-dilution and pre-freezing, sperm progressive motility, plasma membrane integrity and viability was similar (P > 0.05) in extender containing 10% LDLs or the control. However, at post-thaw the aforementioned parameters were higher (P < 0.05) in extender containing 10% LDLs compared with the control and other experimental extenders. The fertility rate of inseminations performed were higher (P < 0.05) with extender containing 10% LDLs than the control. It was concluded that LDLs (10%) in extender improved the freezability and fertility of buffalo bull spermatozoa.     

Analysis of lipid and protein components of ejaculated bull sperm surface and seminal plasma

Surface components of ejaculated bull sperm were radiolabeled by enzymatic iodination with lactoperoxidase and Na¹²⁵I. The sperm were lysed and the labeled components analyzed on SDS-7.5% polyacrylamide gels. Electrophoresis of solubilized radioactivity resolved six components with approximate molecular weights of 77, 61, 44, 36, 24, and 15 kilodaltons. To identify components that might be adsorbed to the bull sperm surface from seminal secretions, seminal plasma was labeled. Electrophoresis of labeled seminal plasma resolved four components with approximate molecular weights of 74, 33, 24, and 15 kilodaltons, each of which comigrated with a labeled sperm surface component. To identify the chemical composition of the radiolabeled components, labeled sperm surface and labeled seminal plasma were submitted to isopycnic density gradient centrifugation in cesium chloride. The ¹²⁵I incorporated into bull sperm surface separated into two discrete areas of radioactivity, one having a density characteristic of protein and the other, of lipid. Iodinated seminal plasma banded in one discrete area that had a density characteristic of protein. Electrophoretic analysis of each area of radioactivity recovered from the gradients demonstrated that five of the six sperm surface and all of the seminal plasma components were in the protein fractions. The 15-kilodalton sperm surface component banded as a lipid, whereas the 15-kilodalton seminal plasma componènt banded as a protein.     

Seminalplasmin,the major basic protein of bull seminal plasma,is a secretory protein of the seminal vesicles

From the experimental results of three independent methods: (1) indirect immunofluorescence employing monospecific anti-seminalplasmin-IgGs, (2) cell-free translation of poly(A)⁺ RNA from seminal vesicle and testicular tissue, as well as (3) Northern analysis of poly(A)⁺ RNA of the latter tissues with a synthetic seminalplasmin-specific antisense DNA probe, it is concluded that the biosynthesis of seminalplasmin occurs in seminal vesicles but not in testis.     

The effect of semen extender,seminal plasma and raw semen on uterine and ovarian blood flow in mares

Transrectal color Doppler sonography was used to evaluate the effect of intrauterine infusion of skim milk semen extender, seminal plasma and raw semen on the endometrium and blood flow in the uterine and ovarian arteries in mares. Six Trotter mares (mean age: 12 years) were examined during estrus in three cycles. Each mare received an intrauterine infusion of 20 ml of skim milk semen extender, seminal plasma or raw semen during estrus in one of three cycles. Blood flow measurements in both uterine and ovarian arteries and the determination of intrauterine fluid via sonography were performed before each infusion and 1, 3, 6, 12, and 24 h after infusion. Forty-eight hours later, the intrauterine infusion and measurements were repeated using the same time intervals. Changes in blood flow were detected using transrectal color Doppler sonography and were evaluated using the mean time-averaged maximum velocity (TAMV) of the blood flow. Cytological and bacteriological examination of uterine swabs performed 48 h after the second infusion revealed less inflammation and bacterial growth in mares infused with skim milk semen extender than in those infused with seminal plasma or raw semen. There was an increase in intrauterine fluid as early as 1 h after infusion of any of the substances. The infusion of skim milk semen extender had no effect on uterine blood flow. Within 1 h after infusion of seminal plasma or raw semen, there was an increase in the TAMV values of both uterine arteries (P<0.05). In contrast, ovarian blood flow increased only in the artery ipsilateral to the preovulatory follicle and only after the infusion of raw semen (P<0.05). In conclusion, the changes in uterine perfusion observed after intrauterine infusion may be associated with endometrial inflammation and vasodilatory components in the seminal plasma, whereas the changes seen in ovarian blood flow are possibly attributable to the interaction between sperm and oviduct.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Effect of low and high egg yolk concentrations in the semen extender for goat semen cryopreservation

This research aimed to evaluate two concentrations of egg yolk inclusion rates (20 and 2.5%) in the semen extender of goat semen cryopreserved during two seasons of the year. The study was conducted during a light-induced breeding season (Experiment 1), and during the natural breeding season (Experiment 2), in the southern hemisphere. Four ejaculates from each buck (n = 2) were collected in each experiment. After collection, semen was divided, with each sample being diluted in the semen extender – according to the treatments (T1 – 20% egg yolk or T2 – 2.5% egg yolk, using a glucose–EDTA extender). For T1 treatment in Experiment 2, the semen was also washed before the semen cryopreservation process. The semen samples were frozen, and after thawing evaluated for seminal characteristics i.e. sperm motility, vigor, morphology and membrane integrity. The fertilising capacity of the frozen-thawed semen was evaluated following a single artificial insemination 12 h after the onset of estrus in 50 (Experiment 1) and 60 does (Experiment 2). In Experiments 1 and 2, the mean values for sperm motility and membrane integrity of the frozen-thawed semen did not differ between the T1 and T2 treatments. However, the mean sperm vigor and morphological normal sperm were greater (P < 0.05) in T2 than T1 treatment. The fertility rates recorded did not differ between T1 and T2 treatments in Experiment 1, however, it was greater (P < 0.05) in the T2 than in the T1 treatment, in Experiment 2. According to obtained results, it can be recommended to use a glucose–EDTA extender with a low egg yolk concentration (2.5%) inclusion, for superior fertility results in goats.     

Effect of butylated hydroxytoluene on cryopreservation of Boer goat semen in Tris egg yolk extender

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.     

Influence of seminal plasma, spermatozoa and semen extender on cytokine expression in the porcine endometrium after insemination

The effects of semen components or extender alone on the expression of selected cytokines [interleukine (IL)-1β, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-10 and transforming growth factor (TGF)-β1] on the porcine endometrium were studied, as well as the presence of polymorphonuclear neutrophilic granulocytes (PMNs). In experiment (Exp) I, groups of gilts were sampled at 5-6h after insemination with fresh semen in extender (Beltsville thawing solution, BTS), spermatozoa in extender (Spz), seminal plasma (SP), or only BTS (control). In Exp II, gilts were sampled 35-40h after insemination with Spz, SP, BTS or only catheter inserted (as control). Immunohistochemical (IHC) labelling of IL-6, IL-10 and TGF-β1 was evident, especially in surface and glandular epithelia of the porcine endometrium. There were no consistent differences in IHC-labelling of the cytokines in relation to different treatments. However, the scores for IL-6 and IL-10 in surface epithelium and sub-epithelial connective tissue compartments were higher at 35-40h than shortly (5-6h) after treatment. Cytoplasmic labelling in the sub-epithelial connective tissue was observed in scattered individual cells but not in PMNs. Shortly (5-6h) after insemination, there were no differences between animals inseminated with BTS (control) and the semen components for any of the cytokine mRNAs. Later however, at 35-40h, lower endometrial expression of TGF-β1 mRNA was observed in the Spz and BTS groups compared with the control (catheter only). The same pattern was found for IL-10 (NS). The mRNA expression of IL-6 in the BTS inseminated group was higher compared to the control group. Insemination with SP resulted in significantly lower PMN cell infiltration in the sub-epithelial connective tissue compared with Spz or BTS groups shortly (5-6h) after insemination. Later (35-40h), a significant difference was found between SP (lower) and the control group (only catheter). To conclude, our results show that insemination and/or inseminated components modulated cytokine expression in the gilt endometrium. The semen extender BTS stimulated immune reactivity, as shown by down-regulation of the suppressive cytokine TGF-β1. Insemination with solely SP clearly decreased PMN cell infiltration of the gilt endometrium. However, no clear relation between the cytokines studied and PMN cell presence was found.     

Effect of glutamine on post-thaw motility of bull spermatozoa after association with LDL (low density lipoproteins) extender: Preliminary results

Glutamine has been used in the composition of semen extenders in several species, but never in the bull. The aim of our study is to demonstrate the cryoprotective role of glutamine for freezing bovine semen and to determine concentration of the latter to improve the motility and trajectory characteristics of spermatozoa. Three experiments were undertaken with 21 ejaculates from three different bulls. In the first experiment, glutamine was added to 40, 80, and 120 mM of basic medium (BM) which consisted of Tris + glycerol 6.4% (v/v). In the second experiment glutamine was added to the 8% low density lipoprotein (LDL) diluent at 40, 80, and 120 mM. In the third experiment, the best concentration of glutamine was determined; this was then added to the LDL extender at 10, 20, 30, and 40 mM. The semen was diluted then frozen in the different media. Motility parameters were assessed using an image analyser following thawing. Experiment 1 demonstrated that glutamine had a cryoprotective effect; at 40 mM it gave superior motility parameters to those obtained with the basic medium (p < 0.05). Experiment 2 demonstrated that the combination of LDL-glutamine 40 mM and 80 mM did not improve motility and even deteriorated it in comparison with the glutamine-free LDL extender. Experiment 3 demonstrated that the addition of 10 mM of glutamine to the LDL medium lead to a significant improvement (p < 0.05) in the motility of bull spermatozoa and could be used to improve bovine semen extenders.     

Influence of seminal plasma on fertility of fresh and frozen-thawed stallion epididymal spermatozoa

The use of epididymal stallion spermatozoa for routine artificial insemination can secure easy future use of valuable genetics after unforeseen death or injury of a valuable stallion.     

Antioxidant effect of quercetin in an extender containing DMA or glycerol on freezing capacity of goat semen

This study was performed to evaluate the effectiveness of quercetin as a non-enzymatic antioxidant in combination with glycerol or Dimethylacetamide (DMA), on freezability of goat semen. Ejaculates from four healthy mature Mahabadi goats were collected using an artificial vagina. After primary processing, semen was pooled and extended by egg yolk based extender supplemented with different concentrations of quercetin (10 or 20 μM) along with 5% glycerol or DMA. The extended semen was frozen and sperm motility parameters, viability, abnormality, membrane integrity and lipid peroxidation were assessed after thawing. Results showed that sperm viability, total motility, progressive motility, straightness (STR) and linearity (LIN) were higher (P < 0.05), and abnormality percentage and MDA concentration were lower (P < 0.05) in extender containing DMA. Similarly, higher (P < 0.05) total motility, progressive motility, viability and membrane integrity along with lower (P < 0.05) MDA level were noted in Q10 group. The lowest (P < 0.05) MDA level was observed in DMA extender containing moderate level of quercetin (Q10D). Also the STR was higher (P < 0.05) in Q10D compared to Q10G and Q20G groups. In conclusion, supplementation of extender with 10 μM quercetin in combination with DMA improves the goat sperm motion kinetics and suppresses lipid peroxidation after freezing and thawing. Furthermore, DMA is more effective cryoprotectant for the freezing of goat sperm.     

Boar seminal plasma or hen's egg yolk decrease the in-vitro chemotactic and phagocytotic activities of neutrophils when co-incubated with boar or bull sperm

The objective was to determine the effects of boar seminal plasma and hen's egg yolk on chemotaxis and phagocytosis of porcine and bovine polymorphonuclear neutrophils (PMNs) in vitro. Chemotactic activity of PMNs was determined following culture for 90 min in a blind well chamber. Phagocytosis was assayed after co-culture of PMNs with sperm for 60 min. In the presence of ≥ 5% boar seminal plasma, chemotactic activity of PMNs was reduced (P < 0.05) in both pigs (from 1126.1 to 934.2-1009.1 cells/mm²) and in cows (from 1067.1 to 768.9-800.0 cells/mm²). Furthermore, ≥ 5% boar seminal plasma reduced (P < 0.05) leukocyte phagocytosis in pigs (26.2-32.1%) and cows (27.2-30.0%) compared to controls (41.7 and 42.1%, respectively). Although 20% hen's egg yolk increased (P < 0.05) chemotactic activity of PMNs in pigs (from 790.4 to 1006.1 cells/mm²) and cows (from 789.9 to 953.5 cells/mm²), egg yolk increased (P < 0.05) phagocytotic activity of porcine PMNs (from 24.3 to 33.8%), but not the activity of bovine PMNs (15.1 vs 15.8% in controls). Boar seminal plasma and caffeine reduced (P < 0.05) the egg yolk-induced increase in chemotaxis in both species (from 988.6 to 795.2 or 813.2 cells/mm² in pigs and from 953.5 to 779.4 or 833.8 cells/mm² in cows), and phagocytotic activities of PMN (from 33.8% to 15.2 or 13.3%) only in pigs (but not in cows; 11.2-15.1%). In conclusion, hen's egg yolk increased chemotactic activity of PMNs in both pigs and cows, whereas egg yolk increased only phagocytosis of PMNs in pigs, but not in cows. Even in the presence of egg yolk, boar seminal plasma and caffeine significantly reduced chemotactic activity of PMNs in pigs and cows, and phagocytotic activity of porcine PMNs.     

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na⁺ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca²⁺ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.     

The retention of 125I-labeled plasma membrane proteins in bovine spermatozoa cryopreserved in egg-yolk citrate extender

Cryopreservation of bovine sperm in egg-yolk citrate extender (EYC) usually maintains fertility. Since plasma membrane proteins are important for the fertilizing potential of sperm, the possible loss of membrane proteins from sperm subjected to cryopreservation in EYC was evaluated. Sperm were washed and labeled with ¹²⁵I without significantly reducing motility. Radiolabeled sperm were a) held for 2 hr at 22°C in N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES)-buffered saline containing 1% polyvinyl alcohol, b) cooled to 5 °C in glycerol-free EYC and held for 3 hr, or c) frozen-thawed in EYC containing 7% glycerol. Sperm were solubilized and proteins were separated by electrophoresis under denaturing conditions. Freeze-thawing dislodged most egg-yolk proteins from the spermatozoal plasma membrane that were bound to and retained by sperm that only were cooled to 5 °C. Autoradiography resolved 11-18 bands of ¹²⁵I polypeptides. There was no difference (P > 0.05) in the amount of ¹²⁵I protein retained by frozenthawed and cooled sperm. However, the radioactivity in two polypeptide bands (MW = 105 K and 24.2 K) was less (P < 0.05) for sperm held at 22 °C in HEPES-buffered saline. Thus, holding sperm in buffered saline at 22 °C resulted in a greater loss of ¹²⁵I proteins from the plasma membrane than did cryopreservation of sperm in EYC. Cryopreservation did not induce greater loss of ¹²⁵I proteins from the plasma membrane than simply cooling sperm to 5 °C in EYC.     

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3–5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.