Effect of low density lipoprotein on the quality of cryopreserved dog semen

Egg yolk is included in extenders for semen cryopreservation due to its protective effect against cold shock, which is attributed to the presence of low density lipoprotein (LDL). This study evaluates how semen quality is affected by using LDL as a replacement for egg yolk in extenders for cooled and frozen dog semen. In Experiment 1, semen was extended in TRIS–glucose at 5 °C, in four treatments: 20% egg yolk (T1); 6% (T2); 8% (T3); and 10% LDL (T4). Sperm motility and membrane integrity after 24, 48, 72 and 96 h and the 50% conservation rate of motile spermatozoa (50 M) were evaluated. The 50 M was less for T1 than for the other treatments (P < 0.01), but T2–T4 did not differ (P > 0.05). In Experiment 2, glycerol at 10% was included in the freezing extender, in treatments similar to those from Experiment 1. Sperm motility and membrane integrity did not differ for T2, T3 and T4 at any period in Experiment 1 and after thawing in Experiment 2 (P > 0.05), but were greater for all LDL treatments than for T1 (P < 0.01), in both experiments. Thus, LDL can replace egg yolk in the composition of the TRIS–glucose extender for cooled or frozen dog semen.     

Effect of sericin supplementation on heat shock protein 70 (HSP70) expression,redox status and post thaw semen quality in goat

Cryopreservation results in substantial deterioration of heat shock protein 70 (HSP70) and ultra-structural changes in sperm organelles, resulting in a marked reduction in post-thaw semen quality. The present study was aimed to explicate the effect of sericin supplementation on expression profile of HSP70, redox status and post-thaw semen quality in Barbari goat. Five Barbari bucks were used to collect thirty semen ejaculates by using artificial vagina and each ejaculate was divided into three aliquots to which sericin was supplemented at 0% (Control), 0.25% (T1) and 0.50% (T2). Further, extended semen samples were equilibrated followed by their cryopreservation. Post-thaw semen characteristics, redox status of seminal plasma, enzyme leakage and HSP70 gene/protein expression in spermatozoa were assessed in all the groups. Per cent progressive motile spermatozoa, spermatozoa having intact plasma membrane (HOST + ve) and intact acrosomes in post-thaw spermatozoa were significantly (p < 0.01) higher in T1 and T2 as compared to control. A significant (p < 0.01) reduction in abnormal spermatozoa was found in T1 as compared to T2. Sericin supplementation significantly (p < 0.05) improved the antioxidative status (SOD, GST, CAT), reduced lipid peroxidation (MDA) and also prevented enzyme (ALT, LDH) leakage as compared to control samples. qRT-PCR results revealed that HSP70 mRNA expression was significantly (p < 0.01) upregulated in T1 and T2 group as compared to control. The positive effect of sericin on expression of HSP70 was further confirmed by immunoblotting followed by densitometry revealing higher expression in T1 and T2 compared to control. Inclusion of 0.25% w/v sericin in semen extender ameliorated the post-thaw semen quality by improving antioxidative status and minimizing the leakage of intracellular enzymes. Sericin supplementation had a beneficial effect on HSP70/HSP70 mRNA expression either by induction or by protection of HSP70/HSP70 mRNA as evident from the gene expression and immunoblotting studies.     

Effects of anti-oxidant additives on microscopic and oxidative parameters of Angora goat semen following the freeze–thawing process

The anti-oxidant system of reduced glutathione (GSH), glutathione peroxidase (GSH-PX), catalase (CAT), and superoxide dismutase (SOD) has been described as a defense functioning mechanism against lipid peroxidation (LPO) in semen, and is important in maintaining sperm motility and viability. This anti-oxidant capacity of sperm cells may be insufficient in preventing LPO during the freeze–thawing process. The aim of this study was thus to determine the influence of varying doses of anti-oxidant additives on standard semen parameters, lipid peroxidation and anti-oxidant activities after the freeze–thawing of goat semen. Ejaculate samples (artificial vagina) obtained from 4 mature Angora goats were evaluated and pooled at 37 °C. The semen samples diluted with a Tris-based extender, containing taurine (25, 50, 75 mM), trehalose (25, 50, 75 mM), and cysteine (5, 10, 15 mM), and an extender containing no anti-oxidant additives (control) were again evaluated. Diluted semen was cooled down to 5 °C and frozen in 0.25 ml French straws, prior to being stored in liquid nitrogen. Frozen straws were thawed in a water bath (37 °C) for 30 s for microscopic sperm evaluation. Upon evaluation of parameters for semen quality, the use of a Tris-based extender supplemented with anti-oxidant additives was found to cause no significant improvement in sperm mortality, when compared to the controls. Increasing doses of taurine and trehalose decreased (P < 0.05) the sperm motility following the freeze–thawing of the goat semen. In biochemical assays, the application of taurine (75 mM) produced the lowest level of malondialdehyde (MDA) (4.46 ± 0.31 nmol/ml), compared to the controls (P < 0.001). Lower GSH levels were higher in the groups in which cysteine was included at 10 and 15 mM (3.27 ± 0.11 and 3.45 ± 0.28 nmol/ml) – compared to the group which received 5 mM cysteine, as well as the controls (2.27 ± 0.08 and 2.50 ± 0.08 nmol/ml respectively, P < 0.001). Compared to the controls, taurine at a concentration of 25 and 75 mM, and increasing doses (50 and 75 mM) of trehalose, significantly increased the GSH-PX activity (P < 0.01). The maintenance of CAT activity was demonstrated to be higher with the addition of 10 and 15 mM cysteine, compared to the other groups (P < 0.001). Vitamin A (VitA) levels were significantly higher, compared to the controls (267.34 ± 9.68 mg/dl and 267.34 ± 9.68 mg/dl, respectively), when 25 mM taurine (329.61 ± 6.35 mg/dl) and 10 mM (318.64 ± 6.34 mg/dl) cysteine was added to the extender (P < 0.001). The results of this study provide a new approach to the cryopreservation of Angora goat semen and could contribute to the improvement of this technology in the goat industry.     

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

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

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls

The aim of this study was to identify different motile sperm subpopulations in ejaculates from an autochthonous bull breed (Bos taurus) and to determine possible modifications in these subpopulations resulting from cryopreservation. Ejaculates were collected and cryopreserved following a conventional protocol. The overall sperm motility and the kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates, after 4 h at 5 °C, and at 0 and 2 h postthaw. A multivariate clustering procedure separated 23,585 motile spermatozoa into four subpopulations: Subpopulation 1 showed medium velocity (VCL: 99.4 ± 17.8 μm/sec) and high progressiveness (LIN: 65.1 ± 14.0%); Subpopulation 2 included spermatozoa with high velocity (VCL: 148.7 ± 25.6 μm/sec) but a nonprogressive trajectory (LIN: 33.1 ± 10.5%); Subpopulation 3 represented slowly motile (VCL: 58.3 ± 24.3 μm/sec) and nonprogressive sperm (LIN: 39.6 ± 18.3%); and Subpopulation 4 included very rapid (VCL: 152.8 ± 25.7 μm/sec) and highly progressive sperm (LIN: 70.9 ± 13.7%). Subpopulation 4 was present in the greatest quantity in fresh ejaculates (36%), but after cooling, it significantly decreased (21%) concomitantly with an increase (P < 0.001) in Subpopulation 2 (from 21% in fresh to 34% in postcooled semen). After freezing and thawing, the overall sperm motility was reduced, mainly due to Subpopulation 2 decreasing from 34% after cooling to 14% after thawing. Differences among bulls in the frequency distribution of spermatozoa within subpopulations were evidenced after thawing by different proportions of spermatozoa in Subpopulations 2 and 4. The current results indicate that a structure of four sperm subpopulations may be a common characteristic of bovine ejaculates and that the cooling phase of cryopreservation seems to be the determinant of postthaw semen quality.     

Cryopreservation of turkey semen by the pellet method: effects of variables such as the extender, cryoprotectant concentration, cooling time and warming temperature on sperm quality determined through principal components analysis

This study was designed to identify the best pellet cryopreservation procedure for the cryosurvival of turkey semen among 192 different treatments established by variations and permutations of seven conditions used in the freezing/thawing process. These conditions were: diluent (IGGKPh, SPh or Tselutin); dilution rate (1:3 vs. 1:4); cooling time (45 vs. 60 min); dimethylacetamide (DMA) concentration as cryoprotectant (6 vs. 8%); equilibration time in DMA (1 vs. 5 min); semen drop volume (50 vs. 80 μL) and thawing temperature (60 vs. 75 °C). Through principal components analysis (PCA), post-thaw sperm quality data (mobility, viability and membrane functional integrity) were reduced to a single output variable (Sperm Quality) indicating overall post-thaw semen quality. All treatments induced a significant reduction in semen quality after warming (P < 0.01), though one set of seven conditions, or treatment, was identified by PCA to generate the highest Sperm Quality score and a further five treatments yielded a score not significantly different (P > 0.05) from this best score. Although still not fulfilling the requirements for commercial application, our findings serve to identify the critical steps in turkey sperm cryopreservation that need to be assessed in future studies.     

Cryostorage of silver barb (Barbodes gonionotus) semen in dry shipper: Efficacy,risk of bacterial cross-contamination and effects of Aeromonas hydrophila on post-thaw sperm quality

This study was conducted to investigate the efficacy of dry shipper for the cryostorage of silver barb (Barbodes gonionotus) sperm, the subsequent risk of bacterial cross-contamination, and the effects of Aeromonas hydrophila on post-thaw sperm. Semen was diluted with calcium-free Hank's balanced salt solution containing 10% ME₂SO, frozen at −8 °C/min and stored for 14 d in a dry shipper. A significant decline (P < 0.05) in the post-thaw sperm motility and viability of samples kept in the dry shipper for 14 d showed a reverse correlation (P < 0.05) with a slight increase in temperature within the dry shipper. The levels of contaminated bacteria in the compartments of the dry shipper were significantly (P < 0.05) lower than those detected in the liquid nitrogen tank. Bacteria from the atmosphere could recontaminate the chambers of the dry shipper and liquid nitrogen tank after 14 d. Bacillus was the most common bacteria isolated from the dry shipper, liquid nitrogen tank, circulating air, bench surface and outer surface of straws. There was no cross-contamination of A. hydrophila from contaminated straws to pathogen-free straws kept in either cryogenic tank. Post-thaw sperm motility and sperm viability significantly (P < 0.05) declined during cryostorage in the dry shipper and liquid nitrogen tank due to the introduction of A. hydrophila and the interaction effect of A. hydrophila and freezing. This study reports, for the first time, the efficacy of a dry shipper for the cryostorage of fish sperm for at least 14 d without a risk of bacterial cross-contamination.     

冷冻对山羊精子转染外源DNA和体外制备转基因胚胎的影响

本实验将鲜精和冻精分别与地高锌标记的线形化的pEGFP-N,质粒孵育转染,用原位杂交方法检测转染效率;PCR和Southern Blotting检测精子与外源DNA的整合效率;与成熟卵母细胞体外受精,PCR检测阳性胚胎比率,用透射电镜技术、碘化丙锭和羟化荧光素双探针技术和单细胞电泳（Single Cell Gel Electrophoresis,SCGE）技术,观察精子冷冻前后的超微结构、精子质膜完整性和精子核DNA损伤的变化,研究冷冻对山羊（Caprahircus）精子转染内化外源DNA和体外制备转基因胚胎的影响及机理。结果表明,冻精显著提高了转染外源DNA的效率（81．60％&#177;16．59％VS32．95％&#177;2．93％,t=4．873,P=0．003;41．80％&#177;6．26％vs27．89％&#177;8．64％,t=2．634,P=0．039）。PCR和Southern Blotting检测表明外源DNA已经整合到精子基因组上。用冻精与成熟卵母细胞体外受精,体外受精穿透率和卵裂率显著低于鲜精组（24．19％&#177;3．15％vs58．86％&#177;3．73％,t=7．131,P〈0．001;11．83％&#177;2．37％vs29．71&#177;3．47％,t=4．302,P〈0．001）,但体外生产的胚胎PCR阳性率比鲜精组显著提高（45．45％&#177;10．87％VS24．44％&#177;6．06％,t=1．750,P=0．013）。超微结构观察和双荧光探针检测都发现冷冻-解冻精子质膜完整性降低（8．34％&#177;4．21％VS65．67％&#177;6．46％,t=12．492,P〈0．001）,SCGE显示冷冻极显著增加了精子彗尾长度和彗星细胞比例（42．67μm&#177;4．56μmvs21．14／Lm&#177;2．36μm,t=5．644,P=0．005;60．00％&#177;4．00％vs17．37％&#177;2．57％;t=15．787,P〈0．001）。冷冻-解冻可以提高山羊精子转染外源DNA的效率,冷冻破坏精子质膜完整性,解除质膜的阻碍作用,是提高外源DNA转染效率的一个主要原因[动物学报54（6）：1089-1097,2008]。     

Effect of daily semen centrifugation and resuspension on the longevity of equine sperm quality following cooled storage

An experiment was conducted to determine whether cooled semen quality could be maintained for a longer interval by conducting daily centrifugation of extended semen, with resuspension of the sperm pellet in fresh extender. Semen treatments included SP10NC and SP50NC which contained 10 and 50% seminal plasma, respectively, were not centrifuged (NC), and were stored at 4 to 7 °C for 96 h. Treatments SP10C and SP50C contained 10 and 50% seminal plasma, respectively, but were centrifuged (C) after 24, 48, and 72 h of cooled storage, with daily resuspension in fresh extender containing 10% seminal plasma. Percent total sperm motility (TMOT) and progressively motile (PMOT) was reduced (P < 0.05) in the SP50NC treatment after 24, 48, 72, and 96 h of storage, and TMOT did not differ (P > 0.05) in the SP10C, SP50C, SP10NC groups after the same storage periods. The % COMP-_αt did not differ (P > 0.05) among treatments at any time period. Percent membrane intact sperm (SMI) was reduced in SP50NC, as compared to SP10C at 48, 72, and 96 h (P < 0.05). Daily centrifugation and resuspension of sperm exposed to 50% seminal plasma for the first 24 h (SP50C) yielded similar TMOT, PMOT, VCL, SMI, % COMP-_αt (P > 0.05) to Groups SP10NC and SP10C after 96 h of storage. Daily centrifugation and resuspension of cool-stored equine semen in fresh extender may be a method to increase sperm longevity.     

Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil

The effects of ambient temperature and humidity, month, age and genotype on sperm production and semen quality in AI bulls in Brazil were evaluated. Data from two consecutive years were analyzed separately. Seven Bos indicus and 11 Bos taurus bulls from one artificial insemination (AI) center were evaluated in Year 1 and 24 B. indicus and 16 B. taurus bulls from three AI centers were evaluated in Year 2. Ambient temperature and humidity did not significantly affect sperm production and semen quality, probably because there was little variation in these variables. Month accounted for less than 2% of the variation in sperm production and semen quality. Increased bull age was associated with decreased sperm motility (P<0.10) and increased minor sperm defects (P<0.001) in Year 1. B. indicus bulls had greater (P<0.005) sperm concentration than B. taurus bulls in both years (1.7×10⁹/ml versus 1.2×10⁹/ml in Year 1 and 1.6×10⁹/ml versus 1.2×10⁹/ml in Year 2, respectively). Ejaculate volume was not significantly affected by genotype in Year 1 (6.6 ml versus 6.9 ml in B. indicus and B. taurus bulls, respectively), but B. indicus bulls had greater (P<0.05) total (11.4×10⁹ versus 8.2×10⁹) and viable (6.7×10⁹ versus 4.9×10⁹) numbers of spermatozoa in the ejaculate than B. taurus bulls. In Year 2, B. taurus bulls had greater (P<0.05) ejaculate volume than B. indicus bulls (8.2 ml versus 6.7 ml, respectively) and total and viable number of spermatozoa in the ejaculate were not significantly different between genotypes (10.3×10⁹ versus 9.1×10⁹ and 6.1×10⁹ versus 5.4×10⁹ in B. indicus and B. taurus bulls, respectively). Sperm motility was not significantly affected by genotype (mean, 59%). In Year 1, B. indicus bulls tended (P<0.10) to have more major sperm defects and had more (P<0.05) total sperm defects than B. taurus bulls (11.8% versus 8.7% and 13.6% versus 10.0%, respectively). In Year 2, B. indicus bulls tended (P<0.10) to have more total sperm defects than B. taurus bulls (16.2% versus 13.3%, respectively). In conclusion, neither ambient temperature and humidity nor month (season) significantly affected sperm production and semen quality. B. indicus bulls had significantly greater sperm concentration and B. taurus bulls had significantly fewer morphologically defective spermatozoa.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen

The objective of this study was to evaluate the effects of reduced glutathione (GSH) and catalase (CAT) supplementation on the kinematics and membrane functionality of sperm during the liquid storage of ram semen, cooled at 5 °C, for up to 24 h. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control) or supplemented with either CAT (100, 200, and 400 U/mL) or GSH (100, 200, and 400 mM) at a final concentration of 50 × 10⁶ sperm/mL. Sperm kinematics, which was analyzed by computer-assisted sperm analysis (CASA), and membrane functionality, which was analyzed using the hypo-osmotic swelling test (HOST), were determined after the addition of the semen samples at different processing times (fresh/diluted, 1.5, 6, 12, and 24 h, at 5 °C). No significant differences were recorded in the kinematics or membrane functionality between treatments at different times. The supplementation of diluents with 100 and 200 U/mL of CAT prevented the harmful effects of cooling on total sperm motility. No significant differences were observed in progressive sperm motility throughout processing, regardless of the treatment and time of evaluation. Supplementation with 400 mM GSH resulted in an earlier reduction (P < 0.05) of total sperm motility, a decrease in rapid sperm rate and a reduction in curvilinear velocity during incubation, at 5 °C. The cooling induced a reduction (P < 0.05) in the percentage of sperm with a functional plasma membrane (HOST), especially after 1.5 h of incubation. Based on the results of the present study, the addition of CAT (100 and 200 U/mL) reduced the deleterious effects of cooling on total motility in ram sperm maintained at 5 °C for 24 h, although it did not affect the functionality of the sperm membranes. However, the addition of 400 mM GSH caused negative effects on the velocity parameters of the sperm.     

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

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

Effects of some thermal, chemical and mechanical treatments on lipase activity in Shammi goat milk

The effects of heating (20, 37 or 50 °C), cooling (5 °C), pasteurisation (71 °C for 15 s), boiling (100 °C), agitation (5 or 10 min), pH (acid or alkaline), and addition of chemicals such as silver and lead nitrates, copper sulphate and sodium chloride on lipase activity in Shammi goat milk were studied. There were non-significant differences (P < 0.01) in chemical composition between Shammi goat milk and Arabi cow milk. Lipase activity in Shammi goat milk was non-significantly (P < 0.01) lower than in Arabi cow milk. Lipase activity in milk of Shammi goats and Arabi cows was reduced when the milk was subjected to heating, cooling, pasteurisation, boiling, or when chemicals or acid was added, whereas in agitated and alkaline milk, the lipase activity was increased. The increase following agitation was greater after 10 min than 5 min. It can be concluded that heating, pasteurising, boiling, cooling, addition of certain chemicals and acidity are means by which lipase activity in milk can be reduced.     

Effects of different concentration and combinations of cryoprotectants on sperm quality,functional integrity in three Indian horse breeds

Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acrosome and DNA integrity as well as other post thaw seminal characteristics of semen of three Indigenous stallion breeds. In the current study, semen was collected from apparently healthy 4–6 years old 3 Marwari, 3 Manipuri and 3 Zanskari breed stallions. After semen collection and evaluation of fresh semen, each semen sample was extended with semen extender containing different concentrations and combinations of Gly and DMF cryoprotectants (i.e. 5% Gly, 5% DMF, 2% Gly, 2% DMF, 2.5% Gly +2.5% DMF and 1% Gly +1% DMF) and frozen. Post thaw semen evaluation was done on the basis of post thaw motility, live sperm count, hypo osmotic swelling test, acrosomal integrity and DNA integrity. Frozen thawed semen showed that the values of plasma membrane integrity, acrosome integrity and DNA integrity parameters were significantly higher (P < 0.05) with 5% DMF than the other cryoprotectants levels and combinations of Gly and DMF. From the present study, it was inferred that the combination of cryoprotectants at different concentrations (Gly and DMF @ 2.5 and 1%) also could not show better enhancement compared to the single cryoprotectant i.e DMF @5% in various post thaw seminal characteristics of Indigenous stallion semen. DMF at 5% concentration gave better protection to the plasma membrane and retained the acrosome and DNA integrity of the spermatozoa. Hence it can be concluded that DMF at 5% can be used for the cryopreservation of the Indigenous stallion with better preservation of the seminal quality.     

The effect of bovine serum albumin and fetal calf serum on sperm quality,DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen

The aim of the present study was to determine the effects of the bovine serum albumin (BSA) and fetal calf serum (FCS) on sperm quality, DNA fragmentation and lipid peroxidation of liquid stored rabbit semen stored up to 72 h at 5 °C. Ejaculates were collected from five New Zealand male rabbits by artificial vagina and pooled at 37 °C following evaluation. Each pooled ejaculate was split into three equal experimental groups and diluted to a final concentration of approximately 40 × 10⁶ sperm/ml (single step dilution), in an Eppendorf tube, with the Tris based extender containing BSA (5 mg/ml), FCS (10%) or no additive (control) at 37 °C, cooled to 5 °C and stored for up to 72 h. The extender supplemented with BSA and FCS did not improve the percentages of motility and acrosomal abnormality during 48 h compared to the control. The additives BSA and FCS had a significant effect in the maintaining of plasma membrane integrity between 48 and 72 h storage period, compared to the control (P < 0.01). The supplementation of BSA and FCS had a protective effect on motility (P < 0.05), plasma membrane integrity (P < 0.01) and acrosomal integrity (P < 0.01) at 72 h compared to the control. The supplementations with BSA and FCS led to a reduction in DNA damage of rabbit sperm at 48 and 72 h during storage period, compared to the control (P < 0.001). Although supplementation of BSA and FCS caused significant (P < 0.01) decreases in malondialdehyde (MDA) level at 48 h and 72 h, they significantly (P < 0.01) increased the glutathione peroxidase (GPx) antioxidant activity up to 72 h when compared to the control group. In conclusion, BSA and FCS supplementation to liquid stored rabbit semen provide a protection for spermatozoa against cool storage-induced DNA damage and plasma membrane integrity by their antioxidative properties.     

The effect of antioxidants on the quality of cryopreserved semen in two salmonid fish, the brook trout (Salvelinus fontinalis) and the rainbow trout (Oncorhynchus mykiss)

Until now the supplementation of cryopreservation extenders with antioxidants has not been examined in teleost fish. Therefore, the present study investigated whether addition of antioxidants (catalase, superoxide dismutase, peroxidase, reduced glutathione, reduced methione, mixtures of reduced and oxidized glutathione or methionine) to the cryopreservation extenders could increase the quality of frozen-thawed semen of brook trout, Salvelinus fontinalis, and rainbow trout, Oncorhynchus mykiss. In brook trout and rainbow trout semen post-thaw fertility and motility were evaluated and in brook trout additionally the membrane integrity, DNA integrity, and sperm lipid peroxidation were evaluated. The tested antioxidants affected the motility parameters, DNA integrity, and fertility of cryopreserved semen, but not the membrane integrity. Most of the observed effects were negative and only minor positive effects were found. In brook trout 1.5 mmol/l reduced methionine and a mixture of 1.5 mmol/l oxidized and reduced glutathione increased the swimming velocity of frozen-thawed semen. One hundred U/l catalase, 1.5 mmol/l reduced glutathione, and 1.5 mmol/l reduced methionine slightly, but not statistically significantly increased the semen post-thaw fertility. However, these effects were not detectable in rainbow trout. Antioxidative stress or damage seems to play no role during cryopreservation, as also in the lipid peroxidation test no differences were obtained between fresh and cryopreserved semen. Therefore, for routine cryopreservation extender supplementation with antioxidants is not recommended in brook trout and rainbow trout.     

The effects of cryopreservation on the acrosome structure,enzyme activity,motility, and fertility of bovine,ovine, and goat sperm

The study was designed to investigate the effects of cryopreservation on bovine, ovine, and goat sperm motility, acrosome structure, enzyme activity, and fertilization ability. Percentage of sperm with hyaluronidase enzyme (HYD) activity was detected by a modified sodium hyaluronate-gelatin membrane. The N-α-benzoyl-DL-arginine-p-nitroanilide (BNPNA) method was used to assess the sperm acrosome enzyme (ACE). The mean percentage of sperm acrosome integrity dropped significantly (P < 0.01) after cryopreservation. The ACE activity of bovine sperm (100.48) was higher (P < 0.01) than that of ovine (57.88) or goat sperm (50.30), while the percentage of sperm with HYD activity of bovine (71.10%) and ovine (67.60%) sperm was higher than that of goat sperm (58.52%) after cryopreservation (P < 0.01). Sperm motility was positively correlated with the activity of the two acrosome enzymes before and after cryopreservation (P < 0.01). Cryopreservation had a negative effect on acrosomal morphology, motility, and acrosomal enzyme activity in their sperm. The fertilization ability of ovine and goat sperm decreased significantly after cryopreservation, but that of frozen bovine sperm did not differ significantly when compared with fresh sperm. There was no significant difference between ovine and goat sperm indices, except for percentage of sperm with HYD activity.     

The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircus ancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities

The aim of the present study was to determine the effects of different doses of raffinose and methionine on post-thawed semen quality, lipid peroxidation and antioxidant enzyme activities of Angora buck (Capra hircus ancryrensis) sperm following cryopreservation.Ejaculates collected from three Angora bucks were evaluated and pooled at 37 °C. Semen samples, which were diluted with a Tris-based extender containing the additives raffinose (2.5, 5, 10 mM) and methionine (2.5, 5, 10 mM) and an extender containing no antioxidants (control), were cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 2.5 and 5 mM methionine led to higher percentages of CASA motility (63.6 ± 7.0; 63.4 ± 3.1%, respectively), in comparison to the controls (P < 0.01) following the freeze-thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective and CASA progressive motilities as well as sperm motion characteristics (VSL and VCL), compared to the control groups (P > 0.05). The freezing extender with raffinose (5 and 10 mM) and methionine at three different doses (2.5, 5 and 10 mM) led to lower percentages of acrosome abnormalities, in comparison to the controls (P < 0.001). In the comet test, raffinose (5 and 10 mM) and methionine (10 mM) gave scores lower than those of the controls, and thereby reduced DNA damage (P < 0.05). Malondialdehyde formation was found to be lower (1.8 ± 0.1 nmol/L) in the group of 5 mM raffinose, compared to the controls following the freeze-thawing process (P < 0.01). The additives did not show any effectiveness on the maintenance of SOD, GSH-PX and GSH activities, when compared to the controls (P > 0.05). In conclusion, methionine and raffinose play a cryoprotective role against sperm CASA motility, acrosome abnormality and DNA damage. Raffinose 5 mM exhibited antioxidative properties, decreasing MDA levels. Further studies are required to obtain more concrete results on the characterization of microscopic parameters and antioxidant activities in cryopreserved goat sperm with different additives.     

Effects of kinetin supplementation on the post-thaw motility,viability, and structural integrity of dog sperm

Oxidative stress is one of the major issues associated with cryopreservation because it causes a marked reduction in the post-thaw quality of semen. This study investigated the ability of kinetin to preserve the structural and functional integrity of dog sperm during cryopreservation. Pooled ejaculates were divided into 5 equal aliquots, diluted with buffer 2 supplemented with different concentrations of kinetin (0, 25, 50, 100, and 200 μM), and finally cryopreserved. The optimal concentration of kinetin was 50 μM based on the significantly improved (P < 0.05) motion characteristics and viability of post-thaw sperm samples. Moreover, kinetin-supplemented samples exhibited significantly higher (P < 0.05) sperm counts with the intact plasma membrane, normal acrosomes, mitochondria, and chromatin than control. The beneficial effects of kinetin were also reflected by the significant increase in the expression levels of anti-apoptotic (B-cell lymphoma, BCL2) and protamine-related genes (protamine 2, PRM2; protamine 3, PRM3), and decrease in the expression of pro-apoptotic (BCL2-associated X, BAX) and mitochondrial reactive oxygen species-modulating genes (ROS modulator 1, ROMO1) in kinetin-supplemented sperm samples than in control. The results demonstrated that supplementation of buffer 2 with 50 μM kinetin is ideal for reducing the magnitude of oxidative damage during semen cryoprocessing and improving the post-thaw quality of dog semen.