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.
This study provides standard information on the attributes of sperm and describes the surface structure of normal and abnormal spermatozoa of Rusa timorensis. Two fertile stags were used as the source of semen collected during the first breeding season commencing from April 5 to July 2, 2012. Another five stags were used as the source of semen collected during the second breeding season commencing from April 1 to June 27, 2013. Semen samples were collected from the stags using an electro-ejaculator. The ejaculate was processed and samples prepared for light and scanning electron microscopy (SEM) according to standard methods. No significant difference (P>0.05) was found between sperm attributes in comparison between different stags and different months of the fertile seasons. The results of this study have also demonstrated that there are no differences in size, shape and surface structure between spermatozoa of the different stags and different months of the fertile seasons. Sperm attributes (volume, pH, sperm concentration, general motility, progressive motility and viability) were 2.2±0.29 ml, 7.2±0.17, 886.3±39.7×10(6) spermatozoa/ml, 78.7±2.01%, 80.8±1.85% and 83.2±0.85%, respectively. Morphological analysis showed low percentage of abnormal spermatozoa 13.9±2.88%. Scanning electron microscopy revealed spermatozoa which consisted of a flat paddle-shaped head, short neck and a tail, which was subdivided into midpiece, principal piece and endpiece. The average spermatozoon was 66.2±0.69 μm in total length. The flat paddle-shaped head was 7.8±0.28 μm long, 4.2±0.15 μm at its widest width, 2.4±0.18 μm basal width and 0.7±0.0 2μm thick. As for the tail, the midpiece length was 13.2±0.14 μm, 0.6±0.04 μm in diameter; the principal piece was 42.6±0.04μm, and 2.8±0.06 μm for the endpiece. Abnormal spermatozoa such as tapered head, microcephalic head, decapitated spermatozoa and bent tails were observed. Results provide standard information useful for development of strategies for semen cryopreservation and assisted reproductive technology in this species.
The semen quality of bucks affects the reproduction performance of the herd and is influenced by genetic and non-genetic factors. Heat shock protein 70 (HSP70) is considered as an important gene affecting semen quality traits. The objectives of this study are to find single nucleotide polymorphisms in HSP70 coding region and their association with semen quality traits on Boer and Boer cross bucks. DNA isolated from 53 goats (36 pure South African Boer and 17 Boer crosses) was subjected to PCR amplification of the exon 1 region of the caprine HSP70 gene. Single-strand conformation polymorphism (SSCP) was used to detect polymorphisms and the variant DNA fragments were sequenced. Two synonymous SNPs (74A>C (ss836187517) and 191C>G (ss836187518)) were detected. Qualities of fresh and post-thaw semen were evaluated for sperm concentration, semen volume, sperm motility and velocity traits, live sperm percentage, and abnormal sperm rate. The C allele of ss836187517 and G allele of ss836187518 were at higher frequencies in both the breeds. The C allele of ss836187517 appeared to be the favorable allele for semen concentration, progressive motility of fresh semen, and motility and sperm lateral head displacement of post-thaw semen. A negative overdominance was observed for ss836187517 alleles on velocity traits of post-thaw semen. The C allele of ss836187518 was favorable for sperm concentration and progressive motility. Results herein suggest that the SNPs in HSP70 may affect on semen quality in tropical regions and specially on the potential of semen for freezing.
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.
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.
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.
Testes from nine male Malin x Santa-Ines rams with an average body weight of 43.1+/-3.53 kg, were used to study the effects of palm kernel cake (PKC) based diet on spermatogenic cells and to assess copper (Cu) levels in liver, testis and plasma in sheep. Animals were divided into three groups and randomly assigned three dietary treatments using restricted randomization of body weight in completely randomized design. The dietary treatments were 60% palm kernel cake plus 40% oil palm frond (PKC), 60% palm kernel cake plus 40% oil palm frond supplemented with 23 mg/kg dry matter of molybdenum as ammonium molybdate [(NH(4))(6)Mo(7)O(24).4H(2)O] and 600 mg/kg dry matter of sulphate as sodium sulphate [Na(2)SO(4)] (PKC-MS) and 60% concentrate of corn-soybean mix+40% oil palm frond (Control), the concentrate was mixed in a ratio of 79% corn, 20% soybean meal and 1% standard mineral mix. The results obtained showed that the number of spermatogonia, spermatocytes, spermatids and Leydig cells were not significantly different among the three treatment groups. However, spermatozoa, Sertoli cells and degenerated cells showed significant changes, which, may be probably due to the Cu content in PKC. Liver and testis Cu levels in the rams under PKC diet was found to be significantly higher (P<0.05) than rams in Control and PKC-MS diets. Plasma Cu levels showed a significant increase (P<0.05) at the end of the experiment as compared to at the beginning of the experiment for PKC and Control. In conclusion, spermatogenesis is normal in rams fed the diet without PKC and PKC supplemented with Mo and S. However spermatogenesis was altered in the PKC based diet probably due to the toxic effects of Cu and the significant changes in organs and plasma. Thus, Mo and S play a major role in reducing the accumulation of Cu in organs.
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.
The Sumatran rhinoceros (Dicerorhinus sumatrensis) is on the verge of extinction in Malaysia. At the Sumatran rhinoceros Conservation Centre in Sungai Dusun, the reproductive behaviour of two female and two male rhinoceroses were studied for 8-10 months during attempts to breed them in captivity. Due to the paucity of scientific information on the reproductive biology of the Sumatran rhinoceros, this study was conducted to obtain information on the reproductive behaviour of this species. The male rhino was introduced to a female rhino in the morning for 1-2 h daily in order to observe for behavioural oestrus. Observations were made on the signs of oestrus and mating behaviour. Oestrus was determined by receptivity towards the male and lasted about 24 h. Common signs of oestrus were an increase in frequency of urine spraying, tail raising or swinging, anogenital and other contacts. Although the males exhibited mounting, the inability of the male to achieve intromission was poor. The study demonstrated that the pattern of courtship and copulation of the captive Sumatran rhinos were comparable with those of other rhino species, reported previously by other scientists and flehmen reflex was also exhibited by the male Sumatran rhinos. In a captive breeding programme, it is recommended that only an oestral female is introduced into a male enclosure due to the male solitary behaviour and to avoid serious injuries inflicted onto the females.
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.
Understanding Macrobrachium rosenbergii ovarian maturation control at the genome level is an important aspect for increasing larvae production. In this study, an ovarian maturation related gene, M. rosenbergii vWD domain and three Kazal-type domains of a gene (MrvWD-Kazal) have been studied. The MrvWD-Kazal gene was isolated using a rapid amplification of cDNA end (RACE) method and the relative abundances of MrvWD-Kazal mRNA in the ovary, hepatopancreas, stomach, intestine and gill were determined by using the quantitative PCR technique. The MrvWD-Kazal gene is composed of 2194 bp with an open reading frame (ORF) of 1998 bp encoding 665 amino acids and has great similarity to the M. nipponense vWD-Kazal gene (91%). The qPCR analyses indicated the relative abundance of MrvWD-Kazal mRNA transcript varied among different stages of ovarian function (P < 0.05), but there were no differences abundance in hepatopancreas, stomach, intestine and gill (P> 0.05). In the ovary, relative abundance of MrvWD-Kazal mRNA transcript gradually increased with ovarian maturation from Stages 1 (Spent; 1.00-fold), to 2 (Proliferative; 3.47-fold) to 3 (Premature; 6.18-fold) and decreased at Stage 4 (Mature; 1.31-fold). Differential relative abundances of MrvWD-Kazal mRNA transcript in the ovary indicate the MrvWD-Kazal protein may have an important function in ovarian maturation of M. rosenbergii. The results of this study also indicate the MrvWD-Kazal is not involved in regulation of the reproductive related function of the hepatopancreas, digestive system (stomach and intestine) and respiratory system (gill).
Testicular volume (TV) is one of the most important traits used in evaluation of the reproductive capacity of male animals. The levelled-container used in the present study was found to be reliable instrument to measure TV, based on a water displacement method. Sperm-associated antigen 11 (SPAG11) is an important gene that affects male reproductive performance. An objective of the present study, therefore, was to determine if single nucleotide polymorphisms (SNPs) in a fragment of the SPAG11 gene could be used to determine associations with values of testicular biometric variables in Boer goats. Primers were designed to amplify the full length of the first two exons of SPAG11. The targeted fragment was generated using a molecular cloning technique. As the result, four SNPs, [g.1256A > G(ss19199134542), g.1270C > T(ss19199134541), g.1325A > G(ss19199134540) and g.1327 G > A (ss19199134543)], were detected using a single-base extension (SBE) method. Two of these SNPs were synonymous (ss19199134540 and ss19199134542). The other two SNPs were nonsynonymous, thus, there were changes in amino acid in the resulting protein: threonine to isoleucine (for ss19199134541) and arginine to glutamine (for ss19199134543). The SNP ss19199134543 was the only locus detected that was associated with TV (P = 0.002). None of the testes dimensions nor TW were associated with detected SPAG11 gene SNPs. Most likely, the ss19199134543 locus affects tissue structures adjacent to the testes, causing the change in TV. In conclusion, among the studied testicular biometric variables, TV had the greatest potential for preselecting of bucks with desirable semen quality. The use of the levelled-container as a TV measurement approach was an accurate and reliable method.
Recently, there has been a growing interest in the ovarian maturation of mud crabs, genus Scylla. Studies regarding the factors that affect ovarian maturation in mud crabs, however, are still lacking. This study, therefore, evaluates the relationship between diet and internal physiological changes of female orange mud crabs, Scylla olivacea. Sixty female adult S. olivacea were sampled from Setiu Wetland, Malaysia. Foreguts were sampled to study fullness and content. The hepatopancreas was sampled to study digestive enzyme activity, biochemical composition, and histology. Ovaries were sampled to study ovarian biochemical composition and histology. Foregut fullness data suggest that there is an increase in feeding with advancing ovarian maturation in mud crabs. Data for foregut contents indicated that when the ovary was in Stage 3 of maturation the diet was animal-based. The activity of amylase, cellulase, lipase, and trypsin during Stages 1 and 4 of ovarian maturation were less than during Stage 3 of ovarian maturation. Biochemical composition (protein and lipid) was greater during Stage 3 of ovarian maturation. Histological analysis of the hepatopancreas indicated an increase in hepatopancreas tubules, B and R cells during Stage 3 as compared with Stage 1 of ovarian maturation. Histological analysis of the ovary indicated increases in oocyte diameter due to concentrated large yolk globules. Based on these results, it is concluded that vitellogenesis mainly occurred during Stage 3 of ovarian maturation, based on evidence of increased feeding with the diet being predominantly animal-based. Likewise, digestive enzyme activities, proteins, lipids, B cells, and R cells were all greater during Stage 3 of ovarian maturation.
The present study examines genetic variation and correlated changes in reproductive performance traits in a red tilapia (Oreochromis spp.) population selected over three generations for improved growth. A total of 328 breeding females (offspring of 111 sires and 118 dams) had measurements of body weight prior to spawning (WBS), number of fry at hatching (NFH), total fry weight (TFW) and number of dead fry (NDF) or mortality of fry including unhatched eggs at hatching (MFH). Restricted maximum likelihood (REML) analysis in a multi-trait model showed that there are heritable genetic components for all traits studied. The heritability for WBS was very high (0.80). The estimates for traits related to fecundity (NFH, TFW) and survival (NDF) were low and they were associated with high standard errors. Genetic correlations of WBS with other reproductive performance traits (NFH, TFW and NDF) were generally positive. However, NFH was negatively correlated genetically with TFW. As expected, body measurements during growth stage exhibited strong positive genetic correlations with WBS. The genetic correlations between body traits and reproductive performance (NFH, TFW, NDF) were not significant. Correlated responses in reproductive traits were measured as changes in least squares means between generations or spawning years. Except for WBS that increased with the selection programs, the phenotypic changes in other reproductive traits observed were not statistically significant (P>0.05). It is concluded that the selection program for red tilapia has resulted in very little changes in reproductive performance of the animals after three generations. However, periodic monitoring of genetic changes in fecundity and fitness related traits such as NDF or MFH should be made in selective breeding programs for red tilapia.
This study describes the fatty acids, total carotenoids, and cell diameter characteristics of the female ovary and hepatopancreas of the mud crab, Scylla olivacea, with comparisons at different ovarian maturation stages. Seventy-one S. olivacea individuals at all stages of ovarian maturation were sampled from the Setiu wetlands, Terengganu, Malaysia. The ovary and hepatopancreas of each crab were used for morphological studies, histological and biochemical analyses (fatty acid composition and total carotenoids). Morphological observations indicated there was an increase in ovarian gonado-somatic index (GSI), with color changes from translucent to dark red; however, a relatively consistent hepato-somatic index (HSI) in the hepatopancreas, with the color ranging from yellow to yellowish-brown. Histological analysis indicated that oocyte diameter was positively correlated with GSI. Hepatopancreatic tubules had a relatively constant diameter from Stage 2 to 4, with increased proportions of R- and B-cells. Biochemical analysis indicated there was a significant increase in total carotenoids in the ovary during maturation. The hepatopancreas, however, had relatively consistent total carotenoid concentrations that were greater than those of the ovary. Overall, the lipid analysis results indicated there were lesser concentrations of fatty acids in the hepatopancreas, while in the ovary there were increasing concentrations during maturation. The lesser concentrations of fatty acids in the hepatopancreas than ovary suggested that energy was transferred to the ovary for future embryonic and larval development. The relationship between the hepatopancreas and the ovary in nutrient content is an important finding in providing a baseline to formulate an optimal diet for improved mud crab hatchery practices.
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.