Tris Extender Research Articles

'Soya milk Tris-based phytoextender reduces apoptosis in cryopreserved buffalo (Bubalus bubalis) spermatozoa'.

The objective of this study was to investigate the effect of newly developed soya milk Tris (SMT)-based phytoextender as an alternative to egg yolk Tris (EYT) extender used for cryopreservation of buffalo (Bubalus bubalis) spermatozoa on apoptosis. Fresh buffalo semen (control without dilution) was cryopreserved in conventional EYT (20% egg yolk v/v in Tris) and SMT (25% soya milk v/v in Tris) extender and used for the assessment of expression of apoptotic proteins. Proteins extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random were separated using SDS-PAGE followed by immunoblotting against caspase-8, caspase-9, caspase-3, poly(ADP-ribose)polymerase (PARP), cytochrome c and apoptosis inducing factor (AIF). In addition, fluorescence microscopy was used for the detection of mitochondrial membrane potential (JC-1 assay) and apoptotic cells (annexin V-FITC/PI assay). The results obtained clearly indicate the significant (p<0.05) reduction in the expression of caspase-3 (27kDa), caspase-8 (53kDa), caspase-9 (50kDa) precursor and cytochrome c (17kDa) in semen cryopreserved in SMT extender in comparison with EYT extender. A non-significant (p>0.05) reduction in expression of PARP-DNA-binding subunit (24kDa) was observed in SMT extender. No expression of AIF was found in cryopreserved semen samples. A significant (p<0.05) increase in the mean percentage of cells having high mitochondrial membrane potential and a non-significant (p>0.05) decrease in late apoptotic cells (AN+/PI+) was observed in SMT extender when compared to EYT extender. The results demonstrated that cryopreservation of buffalo semen in SMT-based phytoextender can replace the traditional egg yolk extenders as it reduces the expression of apoptotic proteins maintaining high mitochondrial membrane potential and gives better protection to sperms in terms of its non-animal origin.

The Quality of Spermatozoa of Gembrong Goats during Cryopreservation Process

Gembrong goat is an Indonesia local goat having specific characteristic that is currently categorized as a breed that is at risk of extinction. In this context, the cryopreservation of gametes is important to support a genome resource bank for storage of gametes for an indefinite period of time. Evaluation of semen and spermatozoa quality was performed to determine the survival of spermatozoa and this information will be used as a reference in the cryopreservation of semen and spermatozoa. The aim of this experiment was to study the characteristics of Gembrong goat’s semen and spermatozoa during cryopreservation process. Once a week, semen from three Gembrong goats (ages about 2-3 years old) was collected using artificial vagina and then frozen with TRIS extender. After freezing, the semen was thawed. Macro- and microscopic parameters of semen and spermatozoa were assessed in fresh and frozen-thawed semen. Results showed that in the fresh semen, the volume was 0.5 mL, sperm abnormalities was 5.74%, sperm concentration was 6731 x 106/mL, the sperm motility was 78.33%, live sperm was 83.17%, and sperm membrane integrity was 78.53%. After-thawing observation showed that sperm motility decreased to 49% (P<0.05) that was lower as compared to that in the fresh and post-equilibration semen. Similarly, the percentage of sperm viability and membrane integrity during cryopreservation showed a similar pattern with the sperm motility. In conclusion, the fresh semen of Gembrong goat had a good quality and met the requirement for further cryopreservation process. Similarly, the quality of frozen-thawed semen of Gembrong goat is eligible for artificial insemination (AI) or in vitro embryo production.

EFFECT OF TREHALOSE, CYSTEINE AND HYPOTAURINE ON BUFFALO BULL SPERM FREEZABILITY, ULTRASTRUCTURE CHANGES AND FERTILIZING POTENTIALS

Cryopreservation induces sublethal damage to the spermatozoa, which leads to reduce their fertile life. The objective of the present study was to investigate the effect of trehalose, cysteine and hypotaurine on freezability, ultrastructure and in vitro fertilizing potentials of the buffalo spermatozoa. Buffalo spermatozoa were cryopresreved with Tris egg yolk extender containing 7% glycerol suplemmented with100 mM trehalose, 100 mM trehalose + 5 mM cysteine, 100 mM trehalose + 25 mM hypotaurine, 100 mM trehalose + 5 mM cysteine + 25 mM hypotaurine or Tris-based extender only (control). Cryopresreved spermatozoa were assessed for post-thawing sperm motility; viability and acrosomal integrity, ultrastructure changes, biochemical activity and in vitro fertilizing potentials. The current results clearly indicated that adding a mixture of 100 mM trehalose , 5 mM cysteine and 25 mM hypotaurine to Tris extender significantly improved (P<0.05) post-thawing sperm motility, viability index and maintained acrosomal integrity following cryopreservation (63.33±7.27%, 155.83±21.06and 12.33±2.73%, respectively) compared with the control spermatozoa (38.33±4.41%, 94.17±12.28 and 25.33±3.49%, respectively). The current results illustrated that addition of combination of additives protected the plasma membrane, acrosomal region and mitochondria and maintained the ultrastructure integrity of the cryopresreved spermatozoa compared with the control spermatozoa. Additionally, the current results revealed that combination of trehalose, cysteine and hypotaurine significantly increased the total antioxidant capacity level (TAC), superoxide dismutase activity (SOD) and glutathione reductase activity (GSH) and reduced significantly the lipid peroxidation rate (0.60±0.89 mµ/ml, 70.00 ±5.78 U/L, 81.66±4.41 U/L and 7.33±1.86 nmol/ml, respectively) compared with the control extender (0.23±0.04 mµ/ml, 34.67±6.74U/L, 51.67±14.82 U/Land 20.67±3.84 nmol/ml, respectively). Furthermore, a mixture of trehalose, cysteine, hypotaurine significantly improved (P<0.05) the rate of in vitro fertilization, cleavage and embryo development to morula and blastocyst stages (62.66±6.28, 53.54±3.89, 23.87±6.28 and17.94±2.57%) compared with the control extender (31.19±4.42, 18.47±5.04, 8.36±1.91 and .42±1.71%, respectively). Therefore, the present results revealed that addition of a mixture of trehalose, cysteine and hypotaurine to the freezing extender could improve semen quality and reduce cryodamage of the buffalo bull spermatozoa.

Comparison of cryoprotective effects of lycopene and cysteamine in different cryoprotectants on bull semen and fertility results.

The objectives of this study were to compare glycerol and ethylene glycol at different concentrations as cryoprotectants and lycopene or cysteamine (with/without) as antioxidants in Tris extender for bull semen. Twenty-four ejaculates were obtained from three bulls. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and added using both of the cysteamine 5mm or lycopene 500μg/ml, and control (without additives). The addition of 7% glycerol with cysteamine, 5% ethylene glycol with cysteamine and 3% ethylene glycol with cysteamine groups gave the lowest CASA motility than the other groups. However, 7% glycerol and 7% glycerol with lycopene resulted in a better rate of CASA progressive motility compared with that of other groups. Generally, all the lycopene groups signed better protective effects on acrosome and total morphology than the other groups. Glycerol 7% and 3% ethylene glycol with lycopene groups yielded to slight higher percentages of membrane integrity assessed by HOST than that of the other groups, but 7% glycerol with cysteamine and 3% ethylene glycol with cysteamine showed the worst percentages of membrane integrity. Glycerol 7% and 5% glycerol with lycopene gave rise to a higher value of VAP, VSL and VCL compared with that of the other groups. On the contrary, adding to 5% glycerol with cysteamine showed negative effect for VAP, VSL, VCL and ALH values. All cryoprotectant groups with lycopene decreased chromatin damage than the other groups. Ethylene glycol 3% led to lower non-return rates of inseminated cows. However, this result was not considered to be statistically important.

IMPACT OF REPLACING EGG YOLK WITH LECITHIN ON SPERM CHARACTERISTICS, BACTERIAL COUNT AND FERTILIZING ABILITY OF CRYOPRESERVED BUFFALO SEMEN.

Soy bean lecithin has been used to replace egg yolk in the semen extender. The aim of this study was to evaluate the effect of replacing egg yolk by soy-lecithin in tris extender on freezability and fertilizing capacity of buffalo semen. Semen from five buffalo bulls was frozen in tris extender containing 0.5, 1.0 and 2% soy-lecithin (treatments) or 15% egg yolk (control). Percentage of motility, livability and intact acrosome spermatozoa and also bacterial count was assessed in diluted, post-equilibrated and post-thawed semen. Results revealed insignificant differences in percentage of sperm motility in diluted semen. However, percentages of sperm motility, livability and intact acrosome spermatozoa increased (P<0.05) and bacterial count decreased (P<0.05) in post-equilibrated and post-thawed semen extended with 0.5% soy-lecithin. These results reflected in the highest conception rate (66.7%) of buffalo cows inseminated with semen containing 05% soy-lecithin compared with 53.3% in semen containing egg yolk (control). In conclusion, egg yolk could be replaced by soy-lecithin in tris-based extender at a level of 0.5% to increase fertilizing capacity of cryopreserved buffalo semen.

Cryoprotection effectiveness of low concentrations of natural and lyophilized LDL (low density lipoproteins) on canine spermatozoa

The aim of this study was to evaluate the use of low concentrations of natural and lyophilized low density lipoprotein (LDL) from hen's egg yolk for cryopreservation of canine semen. Different ammonium sulphate concentrations were tested to extract LDL from egg yolk. The yolk was centrifuged, and LDL was isolated using 10, 20, 40, 45, or 50% ammonium sulphate solution (ASS). The LDL-rich floating fraction was collected for chemical characterization. Dry matter content was lowest (P&lt;0.05) in the LDL extracted with the 50% ASS. The purification of LDL increased in association with increasing ammonium sulphate concentrations. SDS-PAGE showed that the 50% ASS solution yielded a purer fraction of LDL from egg yolk. For semen cryopreservation, TRIS extender was used replacing 20% egg yolk (control) by natural or lyophilized LDL using 1, 2, and 3% (w/v). Semen was centrifuged (755Xg for 7 min), diluted with one of the extenders, packed into 0.5mL straws (100x106 sperm/mL), and placed in a programmable cryopreservation machine. Thawed semen (37°C/ 30s) was analyzed for sperm motility, morphology, and by the hypoosmotic and epifluorescence tests (CFDA/ PI). Natural LDL extracted with 50% ASS was as effective as whole egg yolk to preserve canine frozen sperm when using low concentrations. The lyophilized LDL, mainly in the two higher concentrations tested (2 and 3%), was unsuitable to maintain the effectiveness of the LDL cryoprotective effect on dog sperm.

Comparing ethylene glycol with glycerol and with or without dithiothreitol and sucrose for cryopreservation of bull semen in egg-yolk containing extenders

There are few studies performed for investigating the roles of different ratio and cryoprotectants with dithiothreitol or sucrose on sperm motility characteristics and antioxidant capacities of post-thawed bull spermatozoa. The objectives of this study were to compare glycerol (G) and ethylene glycol (EG) at different concentrations as cryoprotectants and dithiothreitol (D) or sucrose (S) (with/without) as antioxidants in Tris extender for cryopreservation of bull semen. Twenty-four ejaculates obtained from three bulls were included in the study. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and diluted using both of the dithiothreitol 5mM or sucrose 25mM, and control (without additives) was cooled to 4°C and frozen in 0.25-ml French straws. when compared to control, different doses cryoprotectants and antioxidants addition no significantly increased the percentages of post-thaw sperm progressive and motitilities, acrosome abnormality and plasma membrane integrity (P>0.05). However, EG3+S yielded the greatest percentages of the total abnormality (P<0.05). As regard to antioxidant activities G7 and EG5 led to lowest MDA activity with or without D or S but, these results were not supported to the GPx activity (P<0.01). The sperm motion characteristics such as VAP, VCL, ALH and BCF gave significantly different results (P<0.05). When compared the DNA integrity, different doses cryoprotectants without antioxidants addition significantly increased the percentages of the tail intensity and tail moment (P<0.05). There were no significant differences observed in non-return rates among all treatment groups (P>0.05).

TREHALOSE AND A CALCIUM CHELATOR FOR RAM SEMEN CRYOPRESERVATION

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Cryo-survival, cryo-capacitation and oxidative stress assessment of buffalo spermatozoa cryopreserved in new soya milk extender

Egg yolk is most commonly used in semen extenders for cryopreservation but, wide variability in composition and potential risk of xenobiotic contamination has raised questions upon the use of egg yolk. Therefore, the present study was designed to develop a soya milk based phytoextender for buffalo semen cryopreservation. Soya milk was prepared from fresh soya beans in laboratory. Soya milk (25% v/v) and glycerol (6.4% v/v) was added in Tris citrate buffer to prepare Soya Milk Tris (SMT) extender. To improve post-thaw sperm quality, cryo-protectants like trehalose (100mM) or taurine (50mM) was supplemented in SMT extender before semen cryopreservation. Post-thaw quality parameters, degree of cryocapacitation and oxidative stress (lipid peroxidation) of spermatozoa cryopreserved in SMT with or without additives were assessed and compared with Egg Yolk Tris (EYT) extender. SMT extender showed no significant (P>0.05) differences in sperm motility, viability, membrane integrity and acrosome integrity as compared to EYT extender. The degree of cryocapacitation and oxidative damage were found significantly (P<0.05) lower in spermatozoa cryopreserved in SMT extender as compared to EYT extender. Supplementation of additives (trehalose or taurine) in SMT extender significantly (P<0.05) reduced oxidative damage. Thus, soya milk extender (25% soya milk v/v; 6.4% Glycerol in tris citrate buffer) with additives (trehalose or taurine) may substitute conventional egg yolk extender (20% egg yolk v/v; 7% Glycerol in tris citrate buffer) for better cryopreservation of buffalo semen.

Refrigeração do epidídimo canino a 4ºC e recuperação dos espermatozoides epididimários utilizando ACP-106c

Objetivou-se avaliar a qualidade dos espermatozoides recuperados da cauda do epidídimo após a refrigeração do complexo testículo-epidídimo (CTE) de cães usando o diluidor ACP-106c. Foram utilizados 60 cães machos adultos, com peso de 10-20 kg. Após a eutanásia, removeu-se o CTE que foi imerso em solução fisiológica 0,9% e transportado em caixa térmica ao laboratório a 30ºC. Para a refrigeração e recuperação dos espermatozoides epididimários, os 60 pares do CTE foram divididos em 4 grupos, de acordo com o tempo de refrigeração do CTE e posterior recuperação espermática: G0h, G6h, G12h e G18h, em que cada par do CTE permaneceu por zero, seis, doze ou dezoito horas a 4ºC, respectivamente. A recuperação dos espermatozoides da cauda do epidídimo foi realizada pela técnica de flutuação utilizando-se o diluidor ACP-106c ou Tris. Para cada epidídimo foi adicionado 1,0 mL de um dos dois diluidores, pré-aquecidos a 37ºC por 5 minutos. Em seguida foram centrifugados a 800g/5 minutos para remoção dos resíduos celulares. Avaliou-se a morfologia, funcionalidade e motilidade espermática total e progressiva, além de parâmetros obtidos pelo CASA. Os dados foram submetidos à ANOVA seguido do teste de Turkey (P &lt; 0,05). Em todos os parâmetros avaliados, não houve diferença entre os diluidores testados (P&gt;0,05). Os valores de motilidade total nos grupos G0h, G6h, G12h, e G18h para o ACP-106c foram 84,4±7,7; 81,6±11,6; 88,3±6,5 e 69,5±16,9, respectivamente, e para o Tris 85,2±8,7; 77,4±14,3; 79,0±17,8 e 65,4±17,9, respectivamente. Um decréscimo na qualidade espermática foi observado após 18 horas de refrigeração em ambos os diluidores. Dessa forma pode-se concluir que o ACP-106c pode ser utilizado para recuperar os espermatozoides epididimários refrigerados e podem ser viáveis por até 12h de refrigeração.

PENGARUH PENAMBAHAN a-TOKOFEROL PADA MEDIA PENGENCER TRIS KUNING TELUR TERHADAP KUALITAS SEMEN CAIR DOMBA GARUT

The study was conducted to know the influence of a- tocoferrol additional into the egg yolk tris extender medium to the liquid semen quality of “Garut” lamb (Ovis aries). Parameter which had been evaluated i.e. percentages of progressive motile sperm, percentages of viable sperm and percentages of plasma membrane. On the 4 th day evaluation showed that the addition of a- tocoferrol with the dose 0,04 g/100 ml into the egg yolk tris extender produced the highest percentages ofprogressive motile sperm, percentages of viable sperm and percentages of plasma membrane ( 49,0 ± 6,5%; 68,6 ± 4,2% and 59,3 ± 6,5% ) but there were not significantly different from control ( 45,5 ± 5%; 66,0 ± 4,6% and 56,2 ± 5,7% )and the addition of a- tocoferrol with the dose 0,02 g/100 ml ( 49,0 ± 6,5%; 66,6 ± 4,0% and 59,3 ± 2,8% ) respectively. In conclusion, the addition of a-tocoferrol in the egg yolk tris extender medium no significant effect on the liquid semen quality.

Effects of vitamin B12 supplementation on the quality of Ovine spermatozoa

The present study was undertaken to investigate the effect of various levels of vitamin B12 in Tris extender on semen quality of Dallagh rams following the cooling and freeze/thawing process. Semen was collected from six healthy and mature rams with an average body weight of 60.0 ± 5.0 Kg using an electro ejaculator. High quality samples were mixed and diluted in Tris extender supplemented with different concentrations of vitamin B12 (0, 1, 2 and 3 mg/ml). The semen aliquots were cooled and preserved at 5°C and their qualities were evaluated during pre-freezing and then the cooled semen samples were packaged into 0.25 ml straws. Straws were frozen in the vapor of liquid nitrogen, and were then stored at -196ºC. Straws were thawed seven days later and the characteristics of spermatozoa were examined. Results of this study showed that the effect of vitamin B12 on characteristics such as viability, motility, progressive motility and normality of spermatozoa were significant in pre and post freezing conditions (P<0.05). In conclusion, for long term storage of semen of Dallagh rams, we recommend using 2 mg/ml of vitamin B12 in semen extender.

Effects of Skim Milk and Tris Extender on Frozen-Thawed Canine Sperm Morphology

Effects of Skim Milk and Tris Extender on Frozen-Thawed Canine Sperm Morphology

Effect of insulin-like growth factor I on functional parameters of ram cooled-stored spermatozoa

The aim of the study was to examine the effects of insulin-like growth factor I (IGF-I) on ram sperm traits after hypothermic storage. Sperm ejaculates from Lacaune rams were diluted in a Tris extender, pooled, divided into groups of IGF-I doses tested (0, 10, 100 or 200 ng.ml-1) and stored (0-5°C) for 96 h. IGF-I elevated whole sperm motility as measured by a Computer-assisted Sperm Analyser (CASA) system, by 24 h (10 ng.ml-1) and 48 h (200 ng.ml-1) of storage, and by progressive movement on each day of storage. After 72 h the sperm samples were analysed for plasma membrane integrity (peanut agglutinin-fluorescein isothiocyanate), membrane stability (annexin V-Fluos) and apoptosis (Yo-Pro®-1) using fluorescence microscopy. The addition of IGF-I (at 100 or 200 ng.ml-1) reduced the ratio of sperm with disrupted membranes and the ratio of annexin V-labelled sperm. The ratio of apoptotic sperm was reduced by IGF-I given at 10 or 100 ng.ml-1 compared with control. Sperm fertilizing ability, determined at 48 h by an in vitro fertilization (IVF) test on bovine oocytes, was increased by IGF-I given at 100 ng.ml-1 from 47.0 to 67.7%. In conclusion, IGF-I maintained ram sperm functions following cooling storage and its effects were reflected in sperm fertilizing ability in vitro.

Effect of Semen Collection in Tris Extender Supplemented with Hypotaurine and Cysteine on Characteristics of Cooled and Post Thaw Boer Goat Spermatozoa

Effect of Semen Collection in Tris Extender Supplemented with Hypotaurine and Cysteine on Characteristics of Cooled and Post Thaw Boer Goat Spermatozoa

Soybean lecithin-based extender as an alternative for goat sperm cryopreservation

The aim of this study was to evaluate the effect of different concentrations of soybean lecithin (SL) in extenders for sperm goat cryopreservation. Sexually mature male Saanen goats (n=4) were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a skim milk-based extender (control group; CG) or Tris extender supplemented with SL at different concentrations (G1=0.04%, SL G2=0.08% SL and G3=0.16%) for a final concentration of 240×106spermatozoa/mL. The semen samples were packed in straws (0.25mL), frozen using an automated system and stored in liquid nitrogen (−196°C). After thawing (37°C/30s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity, acrosome integrity and mitochondrial activity. No significant difference was observed among the experimental and control groups for all of the parameters (P>0.05). However, even though the control group presented a significantly lower mitochondrial membrane potential compared to fresh semen (P<0.05), the same did not occur for the extender supplemented with soybean lecithin, that is, it did not differ from fresh sperm (P>0.05). The extender containing soybean lecithin at different concentrations preserved the sperm quality parameters in a manner similar to the conventional skim milk-based extender. Thus, it is concluded that an extender containing soybean lecithin as the lipoprotein source can be used for freezing goat semen.

Effect of dietary l-carnitine supplementation on semen characteristics of male Japanese quail

The objective of the present study was to investigate the effects of various levels of dietary l-carnitine supplementation (0, 125, 250, and 500 mg/kg) on quail semen characteristics during liquid storage. Semen was collected using abdominal massage and suitable samples were mixed together, and sperm characteristics including percentage of motile, live, abnormal, semen pH, volume, and concentration were assessed. This experiment was carried out on the basis of completely randomized design. Results showed that the effect of l-carnitine on semen concentration was significant (P 0.05). The effect of l-carnitine on motile and live percentage of sperm in milk and tris extenders in fresh condition was significant (P < 0.05). The highest motile and live sperms were obtained at the 125 mg/kg l-carnitine level. Therefore, use of l-carnitine supplementation (125 mg/kg) in male Japanese quail feeding is recommended to improve semen quality.

Cryopreservation of collared peccaries (Tayassu tajacu) semen using a powdered coconut water (ACP-116c) based extender plus various concentrations of egg yolk and glycerol

The objective was to determine the effectiveness of a powdered coconut water-based extender (ACP-116c), plus various concentrations of egg-yolk and glycerol, as an alternative for cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were apportioned into aliquots that were diluted in Tris plus 10% egg yolk and 3% glycerol, or in ACP-116c plus 10 or 20% egg yolk and 1.5 or 3% glycerol. Samples were frozen in liquid nitrogen and, after 1 mo, thawed at 37 °C for 1 min. After thawing, samples were evaluated as reported for fresh semen, and also for sperm membrane integrity (fluorescent probes) and kinematic parameters (computerized analysis). Results were presented as means ± SEM. Freezing and thawing decreased sperm characteristics relative to fresh semen. Overall, ACP-116c plus 20% egg yolk and 3% glycerol provided better (P < 0.05) sperm motility and kinetic rating (48 ± 6.1% and 2.8 ± 0.2, respectively) after thawing than Tris extender (30.4 ± 5.7% and 2.4 ± 0.2). However, there were no differences (P > 0.05) among treatments with regard to the other sperm characteristics. Based on computerized motion analysis, total (26.5 ± 5.9%) and progressive (8.1 ± 2.2%) motility were best preserved (P < 0.05) with the above-mentioned treatment. In conclusion, a coconut water-based extender, ACP-116c, plus 20% egg yolk and 3% glycerol, was effective for cryopreservation of semen from collared peccaries.

EFECTO DE DOS ANTIOXIDANTES (TEMPO Y TEMPOL) EN LA CRIOPRESERVACIÓN DE SEMEN OVINO EMPLEANDO UN DILUTOR EN BASE A TRIS.

Thirty-two semen samples from four rams were frozed using two antioxidants, Tempo (2,2,6,6 tetramethyl-1-piperidinyloxyl) and Tempol (4-hidroxi 2,2,6,6 tetramethyl-1- piperidinyloxyl), each one in concentrations of 0.5, 1.0, and 2.5 mM, to evaluate the effects on post-thawing motility, viability, acrosomal integrity, and earlier sperm capacitation. Eight replications for each antioxidant concentration and control group, were done using four semen samples per replication. Semen samples were diluted on a Tris extender. Results showed that Tempo 0.5 mM improved frozen semen quality in comparison with control group (p&lt;0.05), by increasing progresive motility (79 vs. 67%), viability and acrosomal integrity (70 vs. 58%), and decreasing earlier sperm capacitation (9 vs. 15%). On the other hand, Tempol decreased frozen semen quality. In conclusion, the use of Tempo 0.5 mM on a Tris extender, at the end of the cooling process, could be an alternative for improving the quality of frozed ram semen.

47 ROLE OF INSULIN-LIKE GROWTH FACTOR-I ON THE FUNCTIONAL PARAMETERS AND FERTILITY OF OVINE FROZEN–THAWED SEMEN

The objective of the present experiment was to evaluate the effects of insulin-like growth factor-I (IGF-I) on the viability and fertility of ovine frozen/thawed semen. For evaluating the semen parameters, 5 rams were used. Before cryopreservation, semen samples from each ejaculate were divided into 4 aliquots and extended with Tris+ alone or supplemented with 1 of 3 different concentrations of IGF-I (50, 100 and 250 ng mL–1). Semen was evaluated immediately after thawing (T0), after 1 (T1) and 2 (T2) post-incubation at 37°C. The percentage of morphologically normal spermatozoa and live cells (fluorescence analysis-calcein and ethidium), acrosome integrity (NAR) and motility were analysed and hypoosmotic swelling tests (HOST) were performed to evaluate in vitro sperm survival. This experiment was replicated 5 times. In addition, AI was performed using 121 ewes to compare the optimal concentration of IGF-I versus Tris+ alone on fertility. Data for sperm parameters were analysed by 1-way ANOVA and the differences among groups were examined by Duncan's multiple range test. Pregnancy rates were analysed by the chi-square test of independence. After 1 and 2 h post-incubation, a decrease was observed in all groups in the percentage of motility, NAR and HOST when compared to the semen at T0. The motility rate was higher (P &lt; 0.05) in the IGF-I 100 and IGF-I 250 groups when compared to the IGF-I 50 and Tris+ alone groups (76.2 and 75% vs 66.2 and 64.4%, respectively) at T0, after 1 h (67 and 63.6% vs 57.4 and 56.2%) and 2 h post-incubation (58.2 and 55.4% vs 48 and 47.2%). Furthermore, viability was higher (P &lt; 0.05) in the IGF-I 100 and IGF-I 250 groups than in the IGF-I 50 and Tris+ alone groups (88.7 and 88.3% vs 76.6 and 77.6%, respectively) at T0. There was no difference (P &gt; 0.05) in NAR or HOST among groups. No significant differences (P &gt; 0.05) in fertility were observed between IGF-I 100 and Tris+ groups. In conclusion, the supplementation of Tris extender with IGF-I (100 and 250 ng mL–1) appears to be a successful way to preserve functional parameters of ovine sperm during cryopreservation.