Y. Rosnina

Effect of sugars on characteristics of Boer goat semen after cryopreservation.

In order to improve Boer goat semen quality during cryopreservation process, the influence of sugar supplementation on semen characteristics of sperm were investigated. Three experiments were carried out to investigate the effect of (a) addition of two monosaccharides (fructose and glucose) and two disaccharides sugars (trehalose and sucrose) (b) sugar combination (fructose and trehalose, sucrose and trehalose, glucose and trehalose), and control (glucose without trehalose) (c) different concentrations of trehalose on cryopreservation using Tris based extender. The total motility, forward motility, viability, normal spermatozoa, acrosome integrity and membrane integrity were assessed subjectively. Differences were not detected among monosaccharides, but glucose increased (P<0.05) sperm forward motility in post-thaw goat semen compared to trehalose or sucrose supplementation. Semen quality did not differ (P>0.05) among disaccharide sugar supplementation. Combination of glucose and trehalose significantly improved the characteristics of Boer spermatozoa after cryopreservation (P<0.05). Supplementation of trehalose (198.24mM) into the glucose extender significantly increased total motility, forward motility, live spermatozoa, acrosome integrity and membrane integrity following cryopreservation (P<0.05). In conclusion, glucose had the better ability to support Boer sperm motility and movement patterns. Combination of monosaccharide (glucose) and disaccharide (trehalose) improved semen quality following cryopreservation. Trehalose supplementation at the concentration of 198.24mM to the glucose extender conferred the greater improvement of semen quality for Boer semen cryopreservation.

Effect of antioxidants on post thaw microscopic, oxidative stress parameter and fertility of Boer goat spermatozoa in Tris egg yolk glycerol extender

This study was conducted to determine the effect of antioxidants on standard semen parameters, lipid peroxidation and fertility of Boer goat semen after cryopreservation. Ejaculates from four bucks were collected, evaluated and pooled at 37°C. The pooled semen was diluted with Tris citric acid fructose for washing. Semen samples, which were diluted with a Tris-based extender containing the antioxidant ascorbic acid (8.5mg/ml), butylated hydroxytoluene (2mM), cysteine (5mM) and hypotaurine (10mM) and an extender without antioxidant supplementation were cooled to 4°C and frozen in 0.25 straws with programmable freezer and finally stored in liquid nitrogen. Data (10 replicates) were analyzed by one-way analysis of variance. Mean (±SEM) progressive motility was significantly higher in ascorbic acid than other supplement groups and control samples (P>0.05). Best values were observed in ascorbic acid followed by BHT, cysteine, and hypotaurine. Antioxidant supplementation in extender showed significant (P<0.05) better values than the control group for sperm membrane integrity, acrosome integrity and viability. The ability of antioxidants to reduce the lipid peroxidation (LPO) after freeze thawing was measured by the formation of malondialdehyde (MDA) using the thiobarbituric acid method. Results showed that addition of antioxidants significantly reduced the rate of LPO in comparison to control (P<0.05). Ascorbic acid exhibited better values (1.27±0.28), than butylated hydroxytoluene, cysteine and hypotaurine 1.32±0.42, 2.27±0.16 and 2.38±0.17 respectively, which are significantly better than control (3.52±0.54). Higher pregnancy rate was observed with ascorbic acid followed by butylated hydroxtolune, hypotaurine and cysteine. However, differences in the fertility rate were non-significant with hypotaurine, cysteine and control groups.

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

The aim of this study was to determine the effect of butylated hydroxytoluene (BHT), a lipid-soluble anti-oxidant added in different concentrations to the Tris egg yolk extenders on semen cytological parameters pre freezing and post thawing (motility, morphology, viability, acrosome integrity and membrane integrity) of Boer goat spermatozoa. A total of 40 ejaculates from four Boer goat bucks were collected using an artificial vagina. Ten replicates of the ejaculates were diluted with a Tris egg yolk based extender which contained various concentrations (0.5mM, 1.0mM, 2.0mM and 3.0mM) of butylated hydroxytoluene while one sample was processed without supplementation of antioxidant and served as control. The diluted semen was cooled at 4°C and loaded into the straw and then stored in liquid nitrogen. It was evident that supplementation of BHT produces positive effect in terms of motility, membrane integrity and acrosome integrity in comparison with the control group in cooled and frozen Boer goat semen. Results showed significant differences in motility, membrane integrity, acrosome integrity and viability of cooled and frozen Boer goat spermatozoa at different concentrations. Motility, membrane integrity, acrosome integrity and viability was significantly higher in all treated groups than the control group (P<0.05) while there was no significant differences (P>0.05) in morphology trait between all group in cooled semen. However, improvement (P<0.05) was observed only in terms of the membrane integrity and acrosome integrity compared to the control and other treated groups in frozen semen. In conclusion, BHT can be used in cryopreservation of Boer goat semen in order to reduce the oxidative stress on spermatozoa.

α-Linolenic acid supplementation in BioXcell® extender can improve the quality of post-cooling and frozen-thawed bovine sperm

The present study was conducted to determine the effects of supplementing α-linolenic acid (ALA) into BioXcell(®) extender on post-cooling, post-thawed bovine spermatozoa and post thawed fatty acid composition. Twenty-four semen samples were collected from three bulls using an electro-ejaculator. Fresh semen samples were evaluated for general motility using computer assisted semen analyzer (CASA) whereas morphology and viability with eosin-nigrosin stain. Semen samples extended into BioXcell(®) were divided into five groups to which 0, 3, 5, 10 and 15 ng/ml of ALA were added, respectively. The treated samples were incubated at 37°C for 15 min for ALA uptake by sperm cells before being cooled for 2 h at 5°C. After evaluation, the cooled samples were packed into 0.25 ml straws and frozen in liquid nitrogen for 24 h before thawing and evaluation for semen quality. Evaluation of cooled and frozen-thawed semen showed that the percentages of all the sperm parameters improved with 5 ng/ml ALA supplement. ALA was higher in all treated groups than control groups than control group. In conclusion, 5 ng/ml ALA supplemented into BioXcell(®) extender improved the cooled and frozen-thawed quality of bull spermatozoa.

Effect of Ascorbic Acid Concentrations, Methods of Cooling and Freezing on Boer Goat Semen Cryopreservation

To improve the Boer goat semen quality during cryopreservation process, three experiments were carried out to investigate the effect of (i) different concentration of ascorbic acid supplementation (ii) rate of cooling with chilled semen characteristics and (iii) method of freezing on post-thaw Boer goat sperm using Tris-based extender. Ascorbic acid at 8.5 mg/ml improved the sperm parameters (motility, integrity of membrane and acrosome, morphology and viability), compared to control in cooled samples (p < 0.05). With regard to other concentrations and post-thawed parameters, ascorbic acid at 2.5-8.5 mg/ml led to higher percentages of sperm motility and integrities of membrane and acrosome when compared to control (p < 0.05). Slow cooling rises to higher percentages of sperm motility, acrosome integrity and viability, in comparison with fast cooling, in terms of cooled and frozen samples (p < 0.05). Programmable freezing method produced the higher percentages of sperm motility, integrities of membrane and acrosome and viability when compared to the freezing method of polystyrene box during goat sperm freezing (p < 0.05). In conclusion, chilled and post-thawed sperm quality of Boer goat was improved when a Tris-based extender supplemented with ascorbic acid was used at stages of different cooling rates and freezing methods.

Alpha-linolenic acid supplementation in tris extender can improve frozen-thawed bull semen quality.

The study was conducted to evaluate the effects of α-linolenic acid (ALA) on frozen-thawed quality and fatty acid composition of bull sperm. For that, twenty-four ejaculates obtained from three bulls were diluted in a Tris extender containing 0 (control), 3, 5, 10 and 15 ng/ml of ALA. Extended semen was incubated at 37°C for 15 min, to allow absorption of ALA by sperm cell membrane. The sample was chilled for 2 h, packed into 0.25-ml straws and frozen in liquid nitrogen for 24 h. Subsequently, straws were thawed and evaluated for total sperm motility (computer-assisted semen analysis), membrane functional integrity (hypo-osmotic swelling test), viability (eosin-nigrosin), fatty acid composition (gas chromatography) and lipid peroxidation (thiobarbituric acid-reactive substances (TBARS)). A higher (p < 0.05) percentage of total sperm motility was observed in ALA groups 5 ng/ml (47.74 ± 07) and 10 ng/ml (44.90 ± 0.7) in comparison with control (34.53 ± 3.0), 3 ng/ml (34.40 ± 2.6) and 15 ng/ml (34.60 ± 2.9). Still, the 5 ng/ml ALA group presented a higher (p < 0.05) percentage of viable sperms (74.13 ± 0.8) and sperms with intact membrane (74.46 ± 09) than all other experimental groups. ALA concentration and lipid peroxidation in post-thawed sperm was higher in all treated groups when compared to the control group. As such, the addition of 5 ng/ml of ALA to Tris extender improved quality of frozen-thawed bull spermatozoa.

Butylated hydroxytoluene can reduce oxidative stress and improve quality of frozen-thawed bull semen processed in lecithin and egg yolk based extenders.

The aims of this study were to evaluate the effects of anti-oxidant butylated hydroxytoluene (BHT), when added at different concentrations into lecithin-based Bioxcell(®) (BX) and two egg-yolk-based; Tris (TY) and citrate (CE) semen extenders, on post-thaw bull sperm quality and oxidative stress. A total of 30 ejaculates from three bulls were collected using an electro ejaculator. Ejaculates were extended with one of the BX, TY and CE extenders, which contained different concentrations (0.0 - control, 0.5, 1.0, 1.5, 2.0 and 3.0mM/ml) of BHT. The extended semen samples were chilled to 4 °C, and then frozen slowly to -196 °C in 0.25 ml straws before being stored in liquid nitrogen for 2 weeks. Results showed that supplementation of BHT improved (P<0.05) general motility, progressive motility, morphology, acrosome integrity, DNA integrity and malondialdehyde of sperm at 0.5mM/ml for BX and at 1-1.5mM/ml of BHT for TY and CE when compared with the control. However, greater concentrations of 2.0 and 3.0mM/ml of BHT had a detrimental (P<0.05) effect compared with the control with all extenders evaluated. In conclusion, BHT supplementation at lesser concentrations (0.5-1.5mM/ml) could improve frozen-thawed bull sperm quality by reducing oxidative stress produced during the freezing-thawing procedures in either lecithin or egg-yolk based extenders.

Effect of different concentrations of egg yolk and virgin coconut oil in Tris-based extenders on chilled and frozen-thawed bull semen

The aim of this study was to evaluate the effects of 8% virgin coconut oil (VCO) combined with different percentages of egg yolk in Tris extender on the quality of chilled and frozen-thawed bull semen. A total of 24 ejaculates from four bulls were collected using an electroejaculator. Semen samples were diluted with 8% VCO in Tris extender which contained different concentrations 0% (control), 4%, 8%, 12%, 16% and 20% egg yolk. The diluted semen samples were divided into two fractions: one was chilled and stored at 4°C until evaluation after 24, 72, and 144h; the second fraction was processed by chilling for 3h at 4°C to equilibrate, then packaged in 0.25ml straws and frozen and stored in liquid nitrogen at -196°C until evaluation after 7 and 14 days. Both chilled and frozen semen samples were then thawed at 37°C and assessed for general motility using computer-assisted semen analysis (CASA), viability, acrosome integrity, and morphology (eosin-nigrosin), membrane integrity (hypo-osmotic swelling test) and lipid peroxidation (thiobarbituric acid-reactive substances (TBARS)). The results indicate treatments with 8%, 12%, 16% and 20% egg yolk with 8% VCO had greater sperm quality (P<0.05) as compared with the control. The treatment with 20% egg yolk had the greatest sperm quality (P<0.05) among the treated groups for both chilled and frozen-thawed semen. In conclusion, the use of 8% VCO combined with 20% egg yolk in a Tris-based extender enhanced the values for chilled and frozen-thawed quality variables of bull sperm.

Plasma progesterone changes and length of oestrous cycle in Rusa Deer (Rusa timorensis).

A study was conducted to profile the plasma progesterone (P4) concentrations and establish the length of oestrous cycle in the Rusa timorensis during the breeding season. Five healthy hinds were selected for peripheral blood sampling twice weekly to gauge the P4 levels by radioimmunoassay, at the start of the breeding season indicated by rutting behaviours of sexually active males. The hinds were polyestrous as proven by the cyclic trend of P4 levels. After the presumptive oestrus indicated by the lowest P4 concentrations (0.20±0.09ng/ml), this ovarian hormone was markedly elevated on day 7 of the cycle (0.78±0.20ng/ml), reached its peak (2.61±0.23ng/ml, P<0.05) on day 14, and then declined to the basal level in the subsequent oestrus. The mean oestrous cycle length in R. timorensis during the breeding season was 19.2 days with a range of 18-21 days, and the pattern of circulating progesterone during the oestrous cycle of the R. timorensis is similar to those of other deer species. It was also observed that the length of oestrous cycle of R. timorensis determined by gauging the progesterone levels and observation of the oestrous behaviours as well as changes in the cellular pattern of vaginal epithelial cells are highly consistent.

Gross anatomy and ultrasonographic images of the reproductive system of the Malayan tapir (Tapirus indicus).

With 4 figures