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1.
The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin‐based semen extender as a substitute for egg yolk‐based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris‐based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post‐thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early‐apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post‐thawed necrotic and late‐apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters.  相似文献   

2.
This study aimed to evaluate various concentrations of egg yolk (5, 10, or 20%) in combination with different concentrations of glycerol (3% or 6%) added to a Tris‐based extender on the post‐thaw characteristics of sperm obtained from Tayassu tajacu. For this purpose, semen from 10 sexually male mature collared peccaries was collected by electroejaculation and evaluated for sperm motility, vigour, viability, morphology and functional membrane integrity. The ejaculates were initially extended in Tris‐fructose plus egg yolk (5%, 10% or 20%). After cooling, the semen was added to Tris‐egg yolk plus glycerol (6% or 12%), resulting in a final concentration of 3% or 6% glycerol of the extender. Straws were frozen using liquid nitrogen and thawed in a water bath at 37°C for 30 s. The frozen–thawed semen was evaluated as reported for fresh semen. After thawing, a significant decrease was verified for sperm motility and vigour, for all the samples in comparison with fresh semen. However, no differences were evidenced among treatments for any sperm characteristics evaluated (p > 0.05), except for the combination between 10% egg yolk and 6% glycerol, which provided the worst preservation of functional membrane integrity (p < 0.05). The interactions between higher concentrations of egg yolk (20%) and glycerol (6%) and also between lower concentrations of the same substances (5% egg yolk and 3% glycerol) added to the Tris‐based extender negatively affected the preservation of the normal sperm morphology after thawing (p < 0.05). In conclusion, the use of Tris‐based extender added to 10% or 20% egg yolk plus 3% glycerol is recommended for effective sperm cryopreservation in collared peccaries.  相似文献   

3.
The study was designed to evaluate AndroMed® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 106 spermatozoa/ml) in tris‐citric egg yolk or AndroMed® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed® extender (45.5% vs. 49%). It is concluded that AndroMed® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.  相似文献   

4.
The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed® and SL‐2; Bioxcell®) and a liposome‐based extender (LS; OptiXcell®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.  相似文献   

5.
Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal‐protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris‐based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5‐ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC‐PNA/PI) and sperm membrane integrity (C‐FDA) were evaluated 5 min post‐thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin‐based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin‐based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin.  相似文献   

6.
Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post‐thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post‐thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili‐Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris–citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 106 spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5‐ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose‐dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome‐intact live sperm (%) and sperm chromatin integrity (%) were better (p < 0.05) in extender having 5.0 ng/ml of arachidic acid compared with control. At 10.0 ng/ml, these values did not vary (p > 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome‐intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post‐thaw quality parameters of cryopreserved Nili‐Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post‐thaw semen quality parameters was observed at 20.0 ng/ml.  相似文献   

7.
This study aimed to investigate the effects of different concentrations of 1,2‐bis‐(o‐aminophenoxy)‐ethane‐N,N,N0 N0‐tetraacetic acid, tetra‐acetoxymethyl ester (BAPTA‐AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing–thawing. After collection, semen was extended (1:1 v/v) on a skim milk‐based extender, centrifuged and resuspended at 400 million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200 μΜ BAPTA‐AM. Motility parameters were assessed after cooling in Equitainer at 5°C for 12, 24, 48, 72 and 120 hr and after freezing–thawing. In addition, mitochondrial membrane potential, intracellular ATP, reactive oxygen species and malondialdehyde concentrations were measured in cryopreserved‐thawed semen. Cooled stored (48 hr) semen containing 50 μΜ BAPTA‐AM and control extender (0 μΜ BAPTA‐AM) was used to assess fertility. Inclusion of 50 μΜ BAPTA‐AM resulted in superior sperm motility parameters during cooled storage when compared to other groups (p < 0.05). Furthermore, semen cryopreserved in extender containing 50 μΜ BAPTA‐AM showed increased intracellular ATP and mitochondrial membrane potential, whereas reactive oxygen species and malondialdehyde were increased after thawing for all groups (p < 0.05). Addition of 50 μΜ BAPTA‐AM to cooling extender resulted in similar pregnancy rates to the control group (75% vs. 73.6%, respectively; p > 0.05). In conclusion, the addition of BAPTA‐AM to semen extenders aided stallion semen cryopreservation in a dose‐dependent manner. Furthermore, the cooling extender supplemented with 50 μΜ BAPTA‐AM could be used to prolong the sperm motility during cooling without apparently compromising fertility. Field trials should be conducted to assess fertility of cryopreserved stallion semen with BAPTA‐AM.  相似文献   

8.
Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN2) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.  相似文献   

9.
The aim of this study was to test and compare two new components in extenders for freezing donkey semen: mare colostrum and jenny colostrum. Colostrum was obtained from four mares and four jennies right after the foal's birth. Ejaculates were collected from five fertile donkeys. Sperm samples were pooled, diluted and cryopreserved in three different experimental extender groups: lactose supplemented with egg yolk extender (20%) as the control group, lactose supplemented with jenny colostrum extender (20%), and lactose supplemented with mare colostrum extender (20%). After thawing, we evaluated the sperm motility by means of computer‐assisted analysis, viability by SYBR‐14 and propidium iodide (PI), membrane functional by HOS test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC‐PNA) and PI. The results demonstrated that lactose–jenny colostrum extender displayed significantly higher values (p < .05) in nearly all parameters evaluated – Total Motility, Viability, HOS test, VCL, VSL, VAP, LIN, STR and WOB –, compared with mare colostrum and egg yolk extenders after thawing. In conclusion, the extender containing jenny colostrum used for donkey semen cryopreservation improved the donkey sperm quality after the freezing–thawing process.  相似文献   

10.
Traditionally, extenders for bull semen included egg yolk or milk, but recently there has been a move to avoid material of animal origin. The aim of this study was to evaluate the effects of two commercial extenders (based on soya lecithin and liposomes) on bull sperm quality after cryopreservation. Post‐thaw sperm quality was evaluated by computer‐assisted sperm analysis and flow cytometric assessment of membrane integrity, chromatin integrity, mitochondrial membrane potential, production of reactive oxygen species and tyrosine phosphorylation. Furthermore, an artificial insemination (AI) trial was conducted, and 56‐day non‐return rates were evaluated. Semen frozen in the liposome‐based extender showed similar membrane integrity and higher mitochondrial membrane potential compared to those in the soya lecithin‐based extender. Chromatin integrity and production of live H2O2+ reactive oxygen species were similar in both extenders. Less superoxide was produced in the samples extended with liposome‐based extender, with or without menadione stimulation. Chromatin integrity and tyrosine phosphorylation were not affected by either type of extender. No differences in 56‐day non‐return rate between extenders containing soya lecithin and liposomes were observed in the AI trial (66% ± 0.8 and 65% ± 0.8, respectively). In conclusion, the sperm quality of bull semen frozen in the two extenders that do not contain material of animal origin was similar, although the semen frozen in the liposome‐based extender had higher mitochondrial membrane potential. Either extender could be used in situations where extenders containing material of animal origin are to be avoided.  相似文献   

11.
Phosphorylation of tyrosine residues on sperm protein is a known indicator of capacitation and a major intracellular signalling event. There is evidence that sperm cryopreservation promotes tyrosine phosphorylation and is associated with reduced fertility of spermatozoa. Under this study, cryoprotective role of different additives namely taurine, trehalose, catalase and 4‐bromophenacyl bromide on buffalo sperm quality was evaluated. Buffalo semen was cryopreserved in tris‐based egg yolk extender supplemented with additives like taurine (50 mm ) or trehalose (100 mm ) or 4‐bromophenacyl bromide (200 μm ) or catalase (100 U/ml) and used for assessment of levels of tyrosine phosphorylation in frozen‐thawed spermatozoa. The results obtained were compared with the level of protein tyrosine phosphorylation of semen cryopreserved in tris‐based egg yolk extender without additives. Proteins were extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random and analysed for tyrosine phospho‐proteins using SDS–PAGE followed by immunoblotting. Monoclonal anti‐phosphotyrosine antibody (Clone pT‐154) was used as primary antibody followed by treatment with HRP‐conjugated secondary antibody. Signals were detected on X‐ray film using chemiluminescence. Nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) were found to be tyrosine phosphorylated in cryopreserved spermatozoa. Supplementation of additives significantly (p<0.05) reduced the level of protein tyrosine phosphorylation in spermatozoa. Moreover, this study showed improved (p<0.05) post‐thaw motility, viability and membrane integrity of spermatozoa on addition of these additives. The results obtained clearly indicate reduced level of capacitation like changes on supplementation of additives in terms of protein tyrosine phosphorylation.  相似文献   

12.
The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post‐thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris‐citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50 mm ) or trehalose (100 mm ), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT‐154 (anti‐phosphotyrosine antibody) and FITC‐conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post‐thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p < 0.05) increased motility, viability and membrane integrity of spermatozoa in both species. Percentage of cryocapacitated spermatozoa was significantly (p < 0.05) higher in cattle as compared to buffalo and degree of cryocapacitaion of spermatozoa decreased significantly (p < 0.05) upon supplementation of additives in both the species. It was also found that tyrosine phosphoproteins were localized differentially in fresh and cryopreserved spermatozoa. Supplementation of taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing–thawing process.  相似文献   

13.
The aim of the present study was to evaluate the effect that the addition of cholesterol‐loaded cyclodextrins (CLC) to the thawing extender has on the quality of frozen‐thawed boar sperm. Pooled semen (n = 5) from three boars was used for the experiments. The semen was cryopreserved with an egg‐yolk‐based extender, it was diluted after thawing in Beltsville thawing solution (BTS) supplemented with different concentrations of CLC (0, 12.5, 25, 50 or 100 mg/500 × 106 sperm), and these samples were incubated at 37°C for 150 min. The following parameters of sperm quality were evaluated 30 and 150 min after incubation: sperm with intact plasma membrane (SIPM; %), sperm with normal acrosomal ridge (NAR; %), total motile sperm (TMS; %), progressively motile sperm (PMS; %) and kinetic parameters. Both SIPM and NAR increased (p < 0.05) when the thawing extender was supplemented with 12.5, 25 and 50 mg CLC/500 × 106 sperm. Nevertheless, motility decreased (p < 0.05) when the concentration of CLC exceeded 12.5 mg CLC/500 × 106 sperm. In conclusion, our results suggest that the supplementation of thawing extenders with CLC improves sperm viability and reduces acrosome damage after freezing/thawing.  相似文献   

14.
The aim of this study was to examine effects of an antibiotic combination at different concentrations on growth of mycoplasma and ureaplasma during cooled storage of canine semen (n = 20). Semen aliquots were diluted with Tris–citric acid–fructose–egg yolk extender containing either 1.0 g/l streptomycin and 0.6 g/l benzylpenicillin (control) or a combination of gentamycin, tylosin, lincomycin and spectinomycin (GTLS‐1: 0.25, 0.05, 0.15 and 0.3; GTLS‐2: 0.5, 0.1, 0.3 and 0.6; GTLS‐3: 1.0, 0.2, 0.6 and 1.2 g/l). Samples were assessed for motility and membrane integrity by computer‐assisted sperm analysis immediately after dilution and at 24, 48 and 72 h of cooled storage. Morphologically, normal spermatozoa were determined, and bacterial culture was performed at 24 and 72 h. Mycoplasma spp. were detected in 14 of 20 ejaculates (70%) with severe growth in 12 samples. A reduction but not total elimination of mycoplasma growth occurred in all GTLS extenders with the most pronounced reduction in group GTLS‐3 (control vs GTLS‐1 and GTLS‐2 p < 0.05, control vs GTLS‐3 p < 0.001). Ureaplasmas were detected in four ejaculates, and growth was reduced to the same extent in GTLS and control extender. Progressive motility in all groups, total motility in groups GTLS 1–3 and percentage of membrane‐intact spermatozoa in groups GTLS 2 and 3 decreased slightly (p < 0.05) over time. In conclusion, dilution of canine semen with GTLS extender has no major detrimental effects on spermatozoa during cooled storage. It reduced the growth but did not totally eliminate mycoplasmas and ureaplasmas from cooled‐stored dog semen.  相似文献   

15.
Cryopreservation of epididymal spermatozoa is often performed after shipping the excised testis–epididymis complexes, under refrigeration, to a specialized laboratory. However, epididymal spermatozoa can be collected immediately after excision of the epididymis and sent extended and refrigerated to a laboratory for cryopreservation. In this experiment, we evaluated the effect of both methods of cold storage bovine epididymal spermatozoa as well as of two different extenders on spermatozoa characteristics after freeze–thawing. For that, spermatozoa collected from the caudae epididymis of 19 bulls were extended and cryopreserved in either AndroMed® or a Tris–egg yolk (TEY)‐based extender. Cryopreservation of sperm cells was performed immediately after castration (Group A, n = 9) or after cold storage for 24 h diluted in the two extenders and (Group B, n = 9) and also after cold storage for 24 h within the whole epididymis (Group C, n = 10). Sperm subjective progressive motility (light microscopy), plasma membrane integrity (hypoosmotic swelling test) and sperm viability (eosin–nigrosin) were evaluated. In vitro fertilization and culture (IVF) was performed to assess the blastocyst rate. No differences (p > 0.05) were observed on post‐thaw sperm parameters between samples from Group A, B and C. TEY extended samples presented a higher (p < 0.01) percentage of progressive motile and live sperm, than those extended in AndroMed®. Blastocyst rate after IVF differed only (p < 0.05) between the reference group (IVF performed with frozen semen with known in vitro fertility) and Group A extended in AndroMed®. We conclude that when cryopreservation facilities are distant from the collection site, bovine epididymal sperm can be shipped chilled overnight either within the epididymal tail or after dilution without deleterious effect on post‐thaw sperm quality. TEY extender was more suitable for cold storage and freezing bovine epididymal sperm, than the commercial extender AndroMed®.  相似文献   

16.
Artificial breeding of mithun poses several challenges including lack of standard protocol for cryopreservation of spermatozoa. This is further complicated by harmful effects of hen's egg yolk (EY) as additive in extender. Purified low‐density lipoproteins (LDL) extracted from EY have been shown as beneficial over EY extender for long‐term semen storage in several species. This investigation explored use of LDL versus EY on semen quality and oxidative stress following freezing–thawing of spermatozoa. A total of 25 of 50 ejaculates based on biophysical parameters were selected for the experiment. After diluting with the Tris‐citrate‐glycerol (TCG) extender, each sample was split into three equal aliquots: Group I, control, EY; Group II and Group III contained 8% and 10% purified LDL, respectively. Frozen–thawed samples were evaluated for motility parameters (progressive, and in the bovine cervical mucus penetration test [BCMPT]), viability, sperm and nuclear abnormality, acrosome integrity, and enzymatic (leakage of intracellular contents) and biochemical (oxidative stress) profiles and in vitro fertility (IVF) assay. Study revealed a significant (p < .05) improvement in viability, sperm and nuclear abnormality, acrosome integrity, motility (progressive and in cervical mucus), cholesterol content, and reduction in the leakage of intracellular enzymes in Group II. Moreover, intactness of acrosome and biochemical membranes was protected significantly (p < .05) in addition to significant (p < .05) improvement in binding per cent and binding index in IVF assay in extender containing 8% LDL. These results demonstrate that although cryopreservation of mithun's spermatozoa in EY was comparable with other species, addition of 8% LDL holds a clear advantage over EY or 10% LDL.  相似文献   

17.
This study evaluated the effect of butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, on dog sperm in chilling storage and cryopreservation. In Experiment 1, 0.2, 0.4, 0.8 and 1.6 mM BHT were added to egg yolk Tris extender (EYT), and sperm were stored at 4°C for 96 hr. Sperm motility, viability, acrosomal integrity and morphological abnormality in the BHT treatment groups were not different from those of the control (0 mM BHT). In Experiment 2, the effect of BHT in EYT containing 0.75% Equex STM paste and 5% glycerol on survivability of cryopreserved sperm was examined after culture at 39°C for 3 hr. Sperm motility, viability and acrosomal integrity in the 0.2 to 0.8 mM BHT treatment groups were not different from those of the control. However, sperm motility, viability and acrosomal integrity decreased when 1.6 mM BHT was added to the extender (P<0.05). In conclusion, supplementation of the extender with 0.2 to 0.8 mM BHT did not affect characteristics of dog sperm in chilling storage and cryopreservation. Supplementation of 1.6 mM BHT did not affect characteristics of chilled sperm but impaired longevity of cryopreserved sperm in the dog.  相似文献   

18.
Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen.  相似文献   

19.
Two experiments were carried out to determine the efficiency of supplementation of ram semen extender with caffeine on chilled storage and frozen capacity of spermatozoa. In the first experiment, eighty ejaculates were collected by an artificial vagina from five adult Barki rams, aged 2–3 years and weighted 45.0 ± 2.0 kg throughout the experimental period (January to February 2017). The ejaculates were pooled and diluted (1:10) with tris‐citric egg yolk extender and were split into five groups. Group 1 served as control, whereas groups 2‐5 were supplemented with 0.1, 0.2, 0.3 and 0.4 mM caffeine. All diluted semen specimens were evaluated for physical characteristics immediately after dilution (T0) and throughout preservation period of 48 hr at 4°C. Simultaneously, oxidative stress and indices such as total antioxidant capacity (TAC), malondialdehyde concentrations (MDA) and alkaline transaminase (AKP) concentrations and value of resazurin reduction test (RRT) were determined. In the second experiment, the raw pooled ejaculates were diluted (1:10) with glycerolated tris‐citric egg yolk extender, receiving the previously mentioned caffeine levels. The post‐thaw assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer‐assisted sperm analysis (CASA) system. The results revealed that adding caffeine to ram semen extender at low (0.1 mM) or medium (0.2 mM) levels had positive impact on both physical characteristics of ram sperm and the enzymatic activities compared to the other semen groups. Caffeine supplementation also enhanced post‐thaw sperm dynamics, which implies its potential as an exogenous antioxidant supplement.  相似文献   

20.
Depending on the mammal species, the use of seminal plasma during semen processing for cryopreservation has been found to have both beneficial and detrimental effects. This study was designed to determine the effects of the second (SF) and third [prostatic fluid, (PF)] ejaculate fractions on plasma membrane and acrosome integrity, mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) translocation and sperm motility in chilled canine spermatozoa by flow cytometry. After pooling the second sperm‐rich fraction of ejaculates from six dogs, samples for each assay were preserved at 5°C for 72 h in egg yolk‐TRIS extender (EYT) alone (control) or supplemented with seminal fluid from the second (EYT‐SF) or third (EYT‐PF) ejaculated fractions. After cold storage, groups EYT‐SF and EYT‐PF showed significantly higher percentages of sperm cells with an intact acrosome [68.8 ± 1.4%, 69.6 ± 2.6% (p < 0.01)] and intact plasma membrane [48.1 ± 2.8%, 50.4 ± 8.2% (p < 0.001)] than that observed in EYT [51.7 ± 3.2% and 33.3 ± 4.1% respectively]. Only in EYT‐SF was PS translocation significantly reduced compared to EYT‐PF and EYT [3.9 ± 0.4%, 10.2 ± 2.2% and 9.0 ± 1.5%, respectively (p < 0.001)]. However, significantly diminished sperm motility was observed in EYT‐SF and EYT‐PF compared to EYT [36.8 ± 2.1%, 35.5 ± 2.3% and 78.4 ± 4.7% (p < 0.001)]. No significant differences were detected in ΔΨm (p > 0.05). In conclusion, supplementing semen extenders with seminal fluid from the second or third fractions of the ejaculate supplementation helps to preserve the integrity of the plasma and acrosome membranes along with the mitochondrial membrane potential but seems to compromise the motility of canine spermatozoa chilled for 72 h.  相似文献   

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