Tris Citric Fructose Research Articles

Evaluation of Motility, Viability and Abnormality of Boar Spermatozoa in Various Modified Extenders

The purpose of this study was to conduct an assessment on the quality of pig semen spermatozoa during storage and determine which diluent material should be used and how long pig semen can be stored. Freshly ejaculated boar semen was obtained from a two-years-old Duroc male and was diluted with Beltsville Thawing Solution (BTS) and Tris Citrate Fructose (TCF) supplemented with 5% egg yolk (KT) and extract of Borassus flabellifer Linn mesocarp (EM) 0.01% and stored at 13°C for up to 4 days. An experimental study with a completely randomized design with four groups and five repeats was applied. Parameters observed were motility, viability and abnormality of spermatozoa. Sperm quality was evaluated by examining motility and abnormality using a phase-contrast microscope with a magnification of 100X, and viability by eosin nigrosine staining. Parametric data were analyzed with ANOVA followed by Duncan test. The results showed that the supplementation of 5% egg yolk in the BTS diluent-based treatment group showed a higher percentage of motility (P<0.05) than without egg yolk, while the TCF-based diluent group showed no differences (P<0.05). Modified-Tris Citrate Fructose diluent supplemented with 5% egg yolk and 0.01% mesocarp extract was able to maintain a significantly higher percentage of viability (67.50±2.0) (P<0.05) than other groups, and had a percentage of abnormalities (9.75±0.957), significantly lower (P<0.05) than other groups. It was concluded that the modification of diluent TCF with 5% egg yolk and mesocarp extract 0.01% provide the best results in maintaining sperm quality with the highest percentage of sperm motility and viability, and the lowest percentage of sperm abnormality compared to the other three diluents. Likewise, all groups of modified-BTS and TCF diluent supplemented with 5% egg yolk and extract of Borassus flabellifer Linn mesocarp in this study were able to maintain motility >40%, viability >45% and abnormalities <20% so they were appropriate for using in the AI procedure.

INRA82 extender enhances semen quality in ram under cooled and cryopreserved stages

Objective: To investigate the effect of INRA82 extender on ram semen quality preserved in cooled and frozen conditions in comparison with Tris-based extenders and to select the suitable extender for ram semen preservation. Methods: Semen samples were collected and pooled from Awassi rams (n=5) and divided into three aliquots. Each aliquot was diluted with one of the following extenders: 1) Tris-citrate glucose, 2) Tris-citrate fructose, and 3) INRA-82. For liquid storage, diluted samples were kept at 5 °C for 72 h. Progressive motility was measured at 0, 24, 48 and 72 h after cooling. Besides, viability, morphology, membrane, acrosome and DNA integrities were evaluated at 24 h post-cooling. For cryopreservation, diluted samples were cooled, equilibrated, loaded and frozen in liquid nitrogen. Thawed samples were evaluated in the same manner as cooled conditions. Results: Seminal characteristics were improved after cooling and thawing in samples diluted with INRA-82 compared to those diluted with Tris-citrate glucose or Tris-citrate fructose (P<0.05). Conclusions: Dilution of ram semen with INRA-82 improves semen parameters. Hence, INRA-82, as a stimulating diluent, can be successfully used to reserve viability and sustainability of cooled and cryopreserved ram semen.

Effect of extender supplemented with date palm pollen grain on bovine semen qualities

Natural extract from plant-based has grown in popularity as protective properties in extender for preserving animal semen. Date palm pollen grain (DPPG) is commercially used for male fertility by enhancing sperm count, motility and DNA quality because DPPG contains flavonoids. Thus, the present study aimed to evaluate the effect of a) extender supplementation with different concentration of DPPG on sperm motility, viability and membrane integrity b) different preservation storage of chilled and frozen bovine semen after seven days. The semen was collected through electrical stimulation and assigned to four treatment groups. The semen were diluted in Tris citric fructose egg yolk (TCFY) diluent (control group; CG) or supplemented with DPPG with different concentration (G1 = 2%, G2 = 4% and G3 = 6% in 20 mg DPPG/40 mL of Tris citric fructose (TCF). Semen samples were chilled (experiment 1) in the refrigerator (4°C) for seven days and cryopreserved in liquid nitrogen (experiment 2) prior to dilution to four treatments. The samples were thawed in a water bath (37 °C) and analysed for motility, membrane integrity and viability by conventional laboratory methods. No significant difference was observed among treatment groups in experiment 1. However, in the second experiment, the addition of 6% DPPG resulted significantly higher (p<0.05) in sperm viability compared to control groups (71.25±1.04) vs (56.47±4.69). The supplementation of 6% DPPG showed the ability to protect the viability of bovine sperm, respectively.

Assessment of Buffalo Semen Preservability Using Tris Extender Enriched With Moringa Oleifera Extract

The aim of the present study was to explore the effect of Moringa oleifera leaves extract (MLE) on buffalo spermatozoa during cooling and cryopreservation. Pooled bull semen were diluted by Tris-Citrate-Fructose (TCF) diluent supplemented with MLE and allocated in 6 concentrations which were 0% control, 10, 20, 30, 40, 50% (v/v) [MLE: TCF] then 20% egg yolk was added, mixed and finally stored at -20°C until used. Diluted semen was cooled slowly up to 5 °C and equilibrated for 4 h. Semen was packed into 0.25 mL polyvinyl straws. After equilibration periods, the straws were exposed to liquid nitrogen (LN2) vapor for 10 minutes and were then dipped in LN2. Extended semen was subjected to evaluation (motility, alive %, abnormality %, and intact sperm membrane (HOST) %) in both chilled and frozen semen. Sperm motility was significantly (P<0.0001) kept high after 11 days of chilling with the concentration 10 and 20%, also it was kept significantly (P<0.0001) high with the concentration 30 and 40% up to 10 days. The use of MEEY had significantly (P<0.0001) improved sperm motility% with the concentration 10 and 20% compared to the control, while the other concentrations didn’t exhibit any effect. On the other hand, MEEY had maintained alive sperm, intact spermatozoa membrane% and abnormal sperm % as good as the control with all the concentrations of MEE used in the present study. Then, it could be concluded that 10-20% of Moringa oleifera leaves extract (under the contemporary conditions of extraction in this study) as a natural additive to semen extenders improved preservability in both chilled and frozen bull semen.

113 Effect of antioxidants on motility and fertility of liquid-stored bovine sperm

It is difficult to maintain sperm in liquid storage for a long time, compared with permanent frozen storage in liquid nitrogen. Antioxidants have been reported to improve the quality and fertility of liquid-stored semen. In this study, we investigated whether antioxidants can extend the motility and fertility of frozen-thawed sperm in liquid storage. Frozen-thawed semen from one Japanese black bull (one ejaculate) was diluted in Tris-citrate-fructose (TCF) diluent with 10% (v/v) egg yolk to a sperm concentration of 1×107 spermmL−1. The antioxidants β-mercaptoethanol (βMe) and glutathione (GSH) were added independently, at various concentrations (0.1, 0.5, 1, and 5mM) to sperm suspensions, and these preparations were compared with Control (no added antioxidant). Sperm suspensions were packaged in centrifuge tubes and placed at 17°C in air and monitored daily until sperm motility had stopped (up to 14 days). Sperm motility was analysed by the Sperm Motility Analysis System (SMAS; Ditect Co. Ltd), and the percentage of progressively motile sperm (straight-line velocity (VSL) of &amp;gt;25μm s−1; Grade A classified by WHO manual), compared with that recorded on Day 0 (100%), was determined each day. For evaluation of fertilizing ability, after incubation in liquid storage for 0, 3, 5, and 7 days, sperm were used for IVF with invitro-matured oocytes (30 oocytes per treatment, three replicates). Embryo development was recorded as the proportion of embryos that reached blastocyst by 8 days after IVF. Data for motility were analysed using one-way ANOVA with Tukey test, and embryo development using chi-squared test. A P-value&amp;lt;0.05 was considered statistically significant. At 7 days, the percentage of progressively motile sperm was significantly higher for 0.5, 1, and 5mM βMe than for Control (30.8%, 48.1%, and 50.3%, vs. 0%, respectively). Treatments with 1 and 5mM βMe maintained some sperm progressive motility for 14 days (9.5% and 14.5%). Treatment with GSH showed the same trend at 7 days (32.2%, 36.3%, and 13.7% for 0.5, 1, and 5mM, vs. 0% for Control); 1 and 5mM GSH maintained sperm progressive motility over 10 days (24.8% and 4.4%). In both antioxidant treatments, embryo development was achieved with sperm stored for up to 5 days (Day 0 vs. Day 5 for 0.1mM βMe: 17.6% vs. 13.8%; for 1.0mM GSH: 26.0% vs. 6.7%; for Control: 17.6% vs. 0%). In this study, antioxidants extended both motility and fertility of frozen-thawed bovine sperm in liquid storage. This result suggests the possibility of application to AI using liquid-stored bovine semen.

Effect of butylated hydroxytoluene on quality of pre—frozen and frozen buffalo semen

Objective: To clarify the antioxidant effect of butylated hydroxytoulene (BHT) at different concentrations on cooled and post frozen semen diluted in tris-citrate-fructose egg yolk glycerol and lecithin -based extenders. Methods: Forty ejaculates were harvested from four buffalo bulls by means of the artificial vagina. Ejaculated semen samples were diluted with each of the tris citrate-fructose egg yolk glycerol and lecithin-based extender diluents. The semen samples diluted with each of the two extenders were added to pre-warmed dried test tubes containing BHT (prepared in ethanol) to get concentrations at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mM/mL BHT. These ingredients were put at 37 °C for 5 min to allow the proper BHT spermatozoal permeation. The diluted semen samples were cooled to 5 °C and then frozen to -196 °C in 0.25 mL ministraws before dipping in liquid nitrogen pending its evaluation. Sperm motility, viability, morphology, intact acrosome and membrane integrity were tested. Visual motility was tested using a high power ordinary microscope (at 400 ×) with closed circuit television, and sperm concentration was tested using Neubauer haemocytometer and abnormality % using eosin-nigrosin stain. Spermatozoal membrane integrity was tested using the hypo-osmotic swelling test. The sperm with swollen twisting tail was normally intact. Sperm acrosomal integrity % was tested as mentioned by Watson. Results: Addition of BHT improved (P<0.01) progressive motility, viability, morphology and acrosome as well as plasma membrane integrities at 0.5-2.0 mM/mL depending upon types of used extenders and stages of pre-and post-freezing process. Higher levels of 2.5 and 3.0 mM/mL BHT had a deteriorating (P<0.01) result if compared to the control and all extenders assayed. Conclusions: BHT addition at lower concentration can improve pre-frozen and post-thawed buffalo sperm quality.

Substitution of egg yolk with different concentrations of soybean lecithin in tris-based extender during bulls' semen preservability

ObjectiveTo investigate the effect of various concentrations of soybean lecithin as an alternative for egg yolk in bull semen extender on post-chilling and post-thaw sperm quality characteristics. MethodsSemen ejaculates were collected from three mature bulls' once/week for 5 weeks. After initial evaluation the approved ejaculates were pooled and extended gradually 1:7 with tris-citrate-fructose egg yolk extender (control) and tris-citrate-fructose (TCF)+different concentration of soybean lecithin (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5% and 4.0%) to ensure 60 million motile spermatozoa/mL, and then proceeded an adopted international cryopreservation protocol. Frozen straws were thawed at 37 °C for 30 s. The parameters studied were sperm motility, viability, abnormality, membrane integrity and normal intact acrosome percentages in chilled and frozen-thawed semen. ResultsThe substitution of egg yolk into TCF with 1% soya lecithin significantly (P < 0.0001) ameliorated the maintenance of semen characters motility (86.67% ± 0.80%), life (87.60% ± 0.88%), and membrane integrity (82.47% ± 0.94%), meanwhile it had significantly (P < 0.0001) reduced the abnormality (13.20% ± 0.60%) of spermatozoa to its least value compared to some other concentrations in use. Moreover, the addition of 1.5% of soya lecithin to TCF had maintained the semen characteristics compared to the control tris-citrate-fructose egg yolk. The significantly higher mean values of post-thawing sperm motility % were observed in 1% soybean lecithin (56.00% ± 1.00%) as compared to the control (41.00% ± 1.00%). Conclusion1–1.5% soya lecithin can effectively alternate egg yolk as cryoprotective additives for cryopreservation extender, without any detrimental effects on post-chilling and post-thaw semen quality in cattle bull.

Chilled and post-thawed semen characteristics of buffalo semen diluted in tris extender enriched with date palm pollen grains (TPG)

The benefits of dates palm and their pollen grains in the human nourishment system have their consideration and importance in medical and nutritional point of view. The present study aimed to investigate a new perspective for the use of date palm pollen grains (DPPG) aqueous extract or infusion in preservation of chilled and frozen buffalo semen. Pooled buffalo semen were diluted by Tris-Citrate-Fructose egg yolk (TCFY) diluent (considered as control) and diverse concentrations of clarified soaked pollen grains in Tris-Citrate-Fructose (TCF) diluent in concentrations of 50, 100, 150, 200 and 250 mg DPPG/5 mL TCF. Five concentrations were kept without egg yolk (TPG) while other five concentrations contained 20% egg yolk (TPGY). Each of the two dilutions was enumerated according to the concentration of DPPG. Extended semen is then chilled (for 7 d) and cryopreserved. Motility, alive sperm and intact sperm membrane (HOST) % were recorded as characteristic sperm parameters. After 7 d of chilling, significant (P < 0.000 1) high percent of motile sperms was recorded for TPG 50, 100, 150, 200 and 250 (66.67, 65.00, 61.67, 61.67 and 63.33, respectively). After freeze thawing, TPGY 150 and 250 showed the significant (P < 0.000 1) high percent of motile sperms (43.75 and 45.00, respectively) while TPGY 100, 200, 250 and TPG 50, 150 revealed the high results concerning HOST (80.61, 83.99, 80.61, 88.33 and 83.33, respectively) whereas the highest alive sperm percentage was recorded with TPGY 150 (96.30) compared to the control. The aqueous cold infusion or extract of the DPPG, added to the TCF extender with or without the addition of egg yolk, proved its good preserving and maintaining capacity of chilled and thawed buffalo bull sperms which was expressed mainly by the sperm motility.

Effect of α-Amylase, Papain, and Spermfluid® treatments on viscosity and semen parameters of dromedary camel ejaculates

Ejaculates from five clinically healthy dromedary camels (Camelus dromedarius) were used to evaluate the effects of different enzymatic treatments (Amylase, Papain, Spermfluid®) on liquefaction and seminal parameters. After collection, ejaculates were divided into 5 aliquots: (1) kept undiluted (control); or diluted 1:1 with: (2) Tris-Citrate-Fructose (TCF), (3) TCF containing Amylase, (4) TCF containing Papain or (5) Spermfluid® containing Bromelain. At 120min after dilution, each aliquot was evaluated, at 20-min intervals, for viscosity, motility, viability and agglutination. Only the aliquots diluted with TCF containing Papain underwent complete liquefaction. Sperm motility decreased significantly during the observation times, except for the samples diluted with Spermfluid® (P=0.005). Diluted samples showed different levels of agglutination, with the lowest being observed in the control and the highest in the Papain-treated samples. The viscosity of dromedary camel ejaculates could be effectively reduced by using the proteolytic enzyme Papain.

Development of an artificial insemination protocol in llamas using cooled semen

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

Acrosomal integrity, viability, and DNA damage of sperm from dasyurid marsupials after freezing or freeze drying

Dasyurids are a diverse group of Australian native carnivores and insectivores that contains several threatened species. Despite successful cryopreservation of sperm from several marsupials, only 3% postthaw motility is reported for dasyurid marsupials. This study examined sperm preservation in the fat-tailed dunnart ( Sminthopsis crassicaudata), an experimental model, with supplementary observations on the eastern quoll ( Dasyurus viverrinus) and northern quoll ( Dasyurus hallucatus). In S. crassicaudata, a toxicity trial demonstrated that incubation with up to 40% glycerol did not reduce sperm viability, suggesting that glycerol is not toxic to dasyurids. On the basis of this finding, S. crassicaudata, D. viverrinus, and D. hallucatus sperm were extended to a final concentration of 20% or 40% glycerol in Tris-citrate fructose and frozen in liquid nitrogen vapor. Postthaw sperm from all three species were nonmotile, and vital staining (SYBR14 and propidium iodide) indicated that sperm were nonviable. However, there was no evidence suggesting disruption of normal gross morphology or loss of acrosomal integrity when assessed by Bryan's staining. After freeze drying, Bryan's staining indicated that approximately 80% of S. crassicaudata sperm had normal acrosomes and no head loss. Despite being nonviable, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling showed that S. crassicaudata sperm frozen in 40% glycerol or freeze-dried had no DNA damage compared with fresh controls. This study has described a method for preservation of the dasyurid sperm nuclei, but continued studies are required to achieve viable motile sperm and establish tools for the long-term storage of dasyurid sperm.

Extender osmolality and sugar supplementation exert a complex effect on the cryopreservation of Iberian red deer ( Cervus elaphus hispanicus) epididymal spermatozoa

We have carried out two experiments to study the cryobiology of red deer epididymal spermatozoa and to improve freezing extenders: (1) effect of extender (Tris-citrate–fructose) osmolality (300–600 mOsm/kg), and (2) effect of sugar (0.4 M) supplementation to the extender (no sugar, glucose, fructose, mannose, sucrose, maltose, threalose and raffinose). Sperm quality was assessed pre-freezing, post-thawing, and after 2 h at 37 °C post-thawing: sperm motility index (SMI), acrosome integrity and membrane integrity (HOS test) were assessed subjectively; mitochondrial activity (JC-1) and membrane stability (merocyanine 540) were assessed by flow cytometry. In experiment 2, DNA status was assessed using acridine orange and flow cytometry. To find an optimal osmolality, we fitted the data to a quadratic curve. Four hundred Osmolal per kilogram rendered better results pre-freezing and post-thawing. However, post-thawing viability and most parameters after 2 h incubation fitted a linear model. Osmolalities above 425 mOsm/kg were deleterious ( P < 0.05). In experiment 2, fructose had a positive effect respect to control after 2 h of incubation at 37 °C post-thawing. Di and trisaccharides were deleterious. Trehalose showed impaired DNA status after 2 h incubation. In conclusion, the osmolality of the extenders should be around 400 mOsm/kg, although the change from quadratic to lineal may indicate a complex effect that must be further studied. Monosaccharides may enhance red deer epididymal sperm cryopreservation, especially fructose, whereas di and trisaccharides may not be adequate.

Effects of Egg Yolk and Cooling Rate on the Survival of Refrigerated Red Deer (Cervus elaphus hispanicus) Epididymal Spermatozoa

Egg yolk is a common component to sperm refrigeration for most of the deer species, the role of which is to protect sperm membranes against cold shock. In addition, there have been many studies of conservation of ejaculated semen from stags, but few have been reported for epididymal spermatozoa. This work was designed to investigate the combined effects of cooling rates (slow: 0.23 degrees C/min vs rapid: 4.2 degrees C/min) from room temperature to 5 degrees C, and egg-yolk concentration (0, 5 or 20%) in the extender on the survival of Iberian red deer epididymal spermatozoa refrigerated at 5 degrees C. Heterospermic sperm samples were diluted to a final sperm concentration approximately 400x10(6) sperm/ml with a Tris-citrate-fructose (TCF)-egg-yolk diluent. Sperm quality was in vitro judged by microscopic assessments of individual sperm motility [sperm motility index (SMI)], and of plasma membrane (hypo-osmotic swelling test) and acrosome (NAR) integrities. Our results first showed that the presence of egg yolk in the extender significantly improves (p=0.01) the viability and sperm motility after sperm dilution. In addition, acrosome and plasma membrane integrities post-refrigeration did not differ significantly between cooling procedures; however, the SMI differed significantly between cooling procedures (slow: 46.6% vs rapid: 50.0%; p=0.01). Our results also showed that sperm quality was significantly (p<0.01) affected by the combined effects of egg-yolk concentration and cooling procedure, being rapid cooling with 20% of egg yolk the most suitable combination for epididymal sperm refrigeration. In conclusion, egg-yolk improved red deer epididymal spermatozoa characteristics after dilution. Rapid cooling protocol using TCF with 20% egg-yolk significantly improved sperm motility of red deer epididymal spermatozoa after cooling.

Studies on the cryopreservation of common wombat (Vombatus ursinus) spermatozoa

In an attempt to develop a gamete-recovery protocol for the northern hairy nosed wombat ( Lasiorhinus krefftii ), spermatozoa were removed from the cauda epididymides of four common wombats ( Vombatus ursinus ) and cryopreserved following a variety of prefreeze storage conditions. Spermatozoa stored for 72 h at 4 degrees C within the testicle before cryopreservation tolerated the freeze-thaw procedure remarkably well, resulting in a higher post-thaw viability (% motile P < 0.01; rate of movement P < 0.01; % live P < 0.01) than sperm recovered on the day of post-mortem, stored in a test tube for 72 h at 4 degrees C and then frozen. The effect of post-thaw dilution with Tris citrate fructose (TCF) diluent on the survival of epididymal common wombat spermatozoa was also investigated. Motility (P < 0.05), rate of sperm movement (P < 0.01) and the percentage of live spermatozoa (P < 0.05) were all significantly greater when spermatozoa were thawed and diluted immediately in TCF than when thawed without dilution. The present study also reports, for the first time, a successful pellet method of freezing wombat spermatozoa on dry ice; volumes of 0.25 and 0.5 mL resulted in higher post-thaw survival compared with 0.1-mL pellets.

Viability and fertility of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15°C

Artificial insemination (AI) in rabbits is not extensive in the breeding programs of the rabbit meat industry. A limiting factor is related to the semen preservation. In order to improve the use of AI, two experiments have been conducted to evaluate sperm viability and fertility of rabbit semen chilled and stored at 15°C after dilution in Tris-based extenders. In Experiment 1, pooled semen samples were diluted 1:10 (semen/extender) in four different Tris-based extenders (Tris-citric-glucose (TCG), TES-Tris-glucose (TTG), Tris-citric-fructose (TCF) and TES-Tris-fructose (TTF)) and stored at 15°C. Sperm viability was evaluated at 0, 24, 48, 72 and 96h following dilution for total sperm motility (TSM), forward progressive motility (FPM), plasma membrane integrity (PMI) and acrosome integrity (NAR). Viability of spermatozoa declined with time of storage (P<0.05), irrespective of the extender used. There were interactions between extender and time of storage (P<0.05) in all viability parameters evaluated. After 96h of storage, TCG provided the highest sperm viability (P<0.05) and TTG the lowest (P>0.05). In Experiment 2, a field trial was conducted at a commercial farm to evaluate the conception and farrowing rates of rabbit spermatozoa extended in TCG. After synchronization of oestrous and induction of ovulation, 3713 does with different physiological conditions (nulliparous, primiparous, lactating and re-breeding) were inseminated one time (15×106 sperm per doses) with semen stored at 0 (n: 1275), 24 (n: 1503) and 48h (n: 935) at 15°C. Overall conception and farrowing rates were 77.1±0.7 and 70.4±0.7, respectively, and the mean litter size was 7.6±0.1. Fertility results were unaffected by the time of semen storage (P>0.05). Regardless of time of semen storage, fertility results were affected by the physiological conditions of does (P<0.05). Nulliparous and lactating does showed the highest fertility and primiparous the lowest. In summary, these results indicate that Tris-buffer extenders are effective for preserving viability and fertilizing capability of rabbit spermatozoa stored at 15°C.