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Fulltext Effects of timed artificial insemination following estrus synchronization in postpartum beef cattle

Malik A, Wahid H, Rosnina Y, Kasim A, Sabri M

Open Vet J, 2012;2(1):1-5.
PMID: 26623282

The objectives of this study were to evaluate estrus response and pregnancy rates resulting from timed artificial insemination (AI) following estrus synchronization using CIDR in postpartum beef cattle. A total of 100 cows were randomly divided into three groups. Groups 1, 2 and 3 were artificially inseminated at 48-50 h (n=30), 53-55 h (n=30) and 58-60 h (n=40) after CIDR removal, respectively. Estrus synchronization was carried out using a CIDR containing 1.38 mg progesterone. All cows were given 2 mg estradiol benzoate, intramuscularly on the day of CIDR insertion (D 0). The CIDR was removed after 8 days and 125 μg of prostaglandin F2α (PGF2α) was injected intramuscularly. One day after CIDR removal all cows were given 1 mg of estradiol benzoate intramuscularly (D 9). Cows were observed visually for estrus after removal of CIDR. Between 30 and 32 days after timed AI, pregnancy was determined using transrectal ultrasonography. The first estrus observation which is approximately 32 h after CIDR removal showed no significant difference (P>0.05) among the three groups. The onset response of estrus after 32 h removal of CIDR was less than 10% in all three groups 6.6% (G1), 6.8% (G2) and 7.3% (G3). Furthermore, percentages of estrus response (D 10) following CIDR removal were 76.6%, 75.0% and 77.5%. The difference between on D 9 and D 10 estrus response were statistically significant (P<0.05). The pregnancy rates were 23.3% (G1), 26.6% (G2) and 37.5% (G3), which were not significant (P>0.05).
Superovulation and egg recovery in goats in the tropics

Rosnina Y, Jainudeen MR, Nihayah M

Vet Rec, 1992 Feb 01;130(5):97-9.
PMID: 1557879

The superovulatory response to gonadotrophin treatment during different months of the year was investigated in Kambing kacang goats, a tropical breed, in Malaysia. Sixty-three cycling does, fitted with progesterone impregnated intravaginal sponges for 17 days, received two days before sponge withdrawal, an intramuscular injection of either 10, 15 or 20 mg of follicle stimulating hormone (FSH) or 500, 1000 or 1500 iu of equine chorionic gonadotrophin (eCG). The dose of FSH was divided into four decreasing daily doses and each daily dose was subdivided into two and administered at 07.00 and 19.00. Fifty-four does detected in oestrus were mated with fertile bucks. The ovarian response was determined by laparoscopy and eggs were recovered surgically five or six days after oestrus. The ovulatory response (mean +/- standard deviation) based on corpora lutea was higher in the FSH (13.4 +/- 8.4 corpora lutea per doe, n = 20) than the eCG-treated groups (6.4 +/- 5.1 corpora lutea per doe, n = 25) but the difference was not significant (P greater than 0.05). Does responded to gonadotrophins throughout the year with more than 50 per cent of does responding during the rainy months compared with less than 35 per cent responding during the dry months. This difference was statistically significant (P less than 0.05). Egg recovery was better in the FSH (6.8 +/- 5.3 per doe, n = 20) than the eCG groups (3.0 +/- 3.8 per doe, n = 21) but the difference was not significant (P greater than 0.05).
Gross anatomy and ultrasonographic images of the reproductive system of the Malayan tapir (Tapirus indicus)

Lilia K, Rosnina Y, Abd Wahid H, Zahari ZZ, Abraham M

Anat Histol Embryol, 2010 Dec;39(6):569-75.
PMID: 20809915 DOI: 10.1111/j.1439-0264.2010.01030.x

The Malayan tapir (Tapirus indicus) is the largest among the four tapir species and is listed as an endangered species. Ultrasound examination and description of the external anatomy of the female reproductive system of three adult females were performed, whereas the internal anatomy was investigated in necropsied samples of four adult females and one subadult female. Descriptions of the male external genitalia were conducted on one adult male. Gross examination revealed the presence of a bicornuate uterus. The uterine cervix is firm and muscular with projections towards its lumen, which is also evident on ultrasonography. The elongated and relatively small ovaries, which have a smooth surface, could not be imaged on ultrasonography, due to their anatomical position. The testes are located inside a slightly pendulous scrotum that is sparsely covered with soft, short hairs. The penis has one dorsal and two lateral penile projections just proximal to the glans penis.
Gross anatomy and ultrasonographic images of the reproductive system of the Sumatran rhinoceros (Dicerorhinus sumatrensis)

Zahari ZZ, Rosnina Y, Wahid H, Jainudeen MR

Anat Histol Embryol, 2002 Dec;31(6):350-4.
PMID: 12693754

The Sumatran rhinoceros (Dicerorhinus sumatrensis) is the smallest of all the rhino species. It is one of the rarest mammals in the world and is in imminent danger of extinction. A study was carried out on seven wild-caught females, three wild-caught males and one captive born female Sumatran rhinoceros at the Sumatran Rhinoceros Breeding Centre in Sungai Dusun, Selangor, Malaysia, beginning 1990. As a result of the paucity of scientific information on the reproductive biology of the Sumatran rhinoceros, this study was conducted to obtain information, which could assist in the captive breeding of this endangered and near extinct species. The anatomy of the reproductive system was based on two post-mortem specimens and transrectal real-time ultrasonography in six adult females. Genitalia of the Sumatran rhinoceros were similar to those of other species of rhinoceroses. The cervix consisted of several folds, the uterus was bicornuate with a short body and prominent horns and the ovaries were completely covered by the fimbriated end of the fallopian tube. The internal genitalia could be imaged by ultrasonography. The testes were located within a pendulous scrotum. Two lateral projections were located at the base of the penis. A well-defined process glandis was present at the tip of the penis. The accessory sex glands and the testes could be imaged by ultrasonography.
Fulltext The nutrient composition and in vitro ruminal degradability of selected local plants used as goat feed in Malaysia

Ghani, A.A.A., Shahudin, M.S., Azrolharith, R., Haizan, A.R., Zamri-Saad, M., Zuki, A.B., et al.

Jurnal Veterinar Malaysia, 2017;29(1):23-25.
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A comparative study of nutrient composition and in vitro ruminal degradability of selected local plants, Macaranga sp.and Mallotus sp.was carried out and Napier grass (Pennisetum purpureum) was used as control.The results obtained from proximate analysis revealed that there were significant difference (P
Alpha-linolenic acid supplementation in tris extender can improve frozen-thawed bull semen quality

Kaka A, Wahid H, Rosnina Y, Yimer N, Khumran AM, Behan AA, et al.

Reprod. Domest. Anim., 2015 Feb;50(1):29-33.
PMID: 25366298 DOI: 10.1111/rda.12445

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.
Effect of ascorbic acid concentrations, methods of cooling and freezing on Boer goat semen cryopreservation

Memon AA, Wahid H, Rosnina Y, Goh YM, Ebrahimi M, Nadia FM

Reprod. Domest. Anim., 2013 Apr;48(2):325-30.
PMID: 22909427 DOI: 10.1111/j.1439-0531.2012.02155.x

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.
Ovarian activity in beef and dairy cows with prolonged postpartum period and heifers that fail to conceive

Yimer N, Rosnina Y, Wahid H, Saharee AA, Yap KC, Ganesamurthi P

Trop Anim Health Prod, 2010 Apr;42(4):607-15.
PMID: 19809886 DOI: 10.1007/s11250-009-9464-y

The primary objectives of this study were to investigate incidence of abnormal ovarian cyclicity (AOC) and its type in dairy and beef cows with prolonged postpartum period (>90 days) and in heifers that fail to conceive. A total of 53 animals were included in the study: 17 Friesian crosses, 16 Braford crosses, eight Brangus crosses, and 12 local Kedah-Kelantan (KKX) crosses. These animals were initially checked for absence of pregnancy via palpation per rectum. Blood samples for progesterone analysis were obtained twice a week for 2 to 3 months following their spontaneous oestrous cycle, and all animals were rechecked for pregnancy at the end of the study. Progesterone analysis indicated that 33.9% of the total animals were having AOC: 18.9% with cessation of ovarian cyclicity, 9.4% with prolonged luteal phases (PLP), and 5.7% short luteal phases. The highest incidence was observed in Brangus crosses (62.5%), followed by Braford crosses (43.8%), and Friesian crosses (35.3%). In contrast, no AOC was observed in the local KKX breeds, and all of them were found to be pregnant at the end of the study. A significant difference (p < 0.05) in the incidence of AOC and its type was observed between Kedah-Kelantan crosses and the other breeds. Although not significant (p > 0.05), Friesian crosses showed a higher percentage incidence of AOC than beef cows (40% vs 36.4%), with major types being PLP (26.7%) in dairy and cessation of ovarian cycle (27.3%) in beef cows. Compared with beef heifers, beef cows showed a higher percentage of AOC (36.4% vs 28.6%) where again, cessation of cyclicity was the predominant abnormality. In conclusion, AOC reflected by abnormal endocrine pattern is a possible cause of reduction in fertility for dairy and beef cows beyond 90 days postpartum and heifers that fail to conceive.
Effect of antioxidants on post thaw microscopic, oxidative stress parameter and fertility of Boer goat spermatozoa in Tris egg yolk glycerol extender

Memon AA, Wahid H, Rosnina Y, Goh YM, Ebrahimi M, Nadia FM

Anim. Reprod. Sci., 2012 Dec;136(1-2):55-60.
PMID: 23182473 DOI: 10.1016/j.anireprosci.2012.10.020

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.
Reproductive behaviour of captive Sumatran rhinoceros (Dicerorhinus sumatrensis)

Zainal Zahari Z, Rosnina Y, Wahid H, Yap KC, Jainudeen MR

Anim. Reprod. Sci., 2005 Feb;85(3-4):327-35.
PMID: 15581515

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

Kaka A, Wahid H, Rosnina Y, Yimer N, Khumran AM, Sarsaifi K, et al.

Anim. Reprod. Sci., 2015 Feb;153:1-7.
PMID: 25544152 DOI: 10.1016/j.anireprosci.2014.12.001

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.
Sperm attributes and morphology on Rusa timorensis: light and scanning electron microscopy

Mahre MB, Wahid H, Rosnina Y, Jesse FF, Azlan CA, Khumran AM, et al.

Anim. Reprod. Sci., 2014 Aug;148(3-4):245-50.
PMID: 25015846 DOI: 10.1016/j.anireprosci.2014.06.006

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.
Effect of semen collection methods on the quality of pre- and post-thawed Bali cattle (Bos javanicus) spermatozoa

Sarsaifi K, Rosnina Y, Ariff MO, Wahid H, Hani H, Yimer N, et al.

Reprod. Domest. Anim., 2013 Dec;48(6):1006-12.
PMID: 23808560 DOI: 10.1111/rda.12206

This study was conducted to evaluate the response of Bali bulls (Bos javanicus) to different semen collection methods and their effects on fresh and post-thawed semen quality. The collection methods employed were electro-ejaculation (EE), transrectal massage (RM) and RM followed by EE (RM + EE). A total of 25 untrained Bali bulls (age between 2 and 4 years old) were subjected to the different semen collection methods. Fresh semen samples from all the 25 bulls were evaluated for volume, pH, general motility, live/dead ratio and abnormality using the conventional method. For fresh and frozen samples collected by EE and RM from 10 bulls, computer-assisted semen analysis system was used for precise quantitative measurement of motility, velocity and forward progression. Accucell photometer was used to measure sperm concentration in all samples, regardless fresh and frozen. Semen samples were obtained 100% of the attempts using EE, 84% using RM and 96% using RM + EE. There were no differences among the collection methods for fresh semen quality characteristics, including motility, morphology and viability, but pH and volume were higher for EE than RM and RM + EE. Higher sperm concentration was observed in semen collected by RM than the other two methods. Different age groups (2-3 and >3-4 years old) of the bulls did not show significant differences in volume, pH, sperm concentration, percentages in motility, live/dead ratio and normal sperm morphology. The quality of semen for general and progressive motility, VAP, VSL and VCL and acrosomal integrity after thawing was higher for RM than EE. In conclusion, Bali bulls appeared to respond best to EE and the combination of RM + EE than RM, as a method of semen collection, with a shorter time of stimulation required. Differences in age of the Bali bulls did not affect the semen quality.
Plasma progesterone changes and length of oestrous cycle in Rusa Deer (Rusa timorensis)

Mahre MB, Wahid H, Rosnina Y, Jesse FF, Azlan CA, Yap KC

Anim. Reprod. Sci., 2013 Oct;141(3-4):148-53.
PMID: 24012278 DOI: 10.1016/j.anireprosci.2013.07.012

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

Memon AA, Wahid H, Rosnina Y, Goh YM, Ebrahimi M, Nadia FM, et al.

Anim. Reprod. Sci., 2011 Nov;129(1-2):44-9.
PMID: 22024366 DOI: 10.1016/j.anireprosci.2011.10.004

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

Naing SW, Wahid H, Mohd Azam K, Rosnina Y, Zuki AB, Kazhal S, et al.

Anim. Reprod. Sci., 2010 Oct;122(1-2):23-8.
PMID: 20637550 DOI: 10.1016/j.anireprosci.2010.06.006

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

Khumran AM, Yimer N, Rosnina Y, Ariff MO, Wahid H, Kaka A, et al.

Anim. Reprod. Sci., 2015 Dec;163:128-34.
PMID: 26515370 DOI: 10.1016/j.anireprosci.2015.10.007

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.
Fulltext Effect of different concentrations of soybean lecithin and virgin coconut oil in Tris-based extender on the quality of chilled and frozen-thawed bull semen

Tarig AA, Wahid H, Rosnina Y, Yimer N, Goh YM, Baiee FH, et al.

Vet World, 2017 Jun;10(6):672-678.
PMID: 28717321 DOI: 10.14202/vetworld.2017.672-678

AIM: The objective of this study was to evaluate the effects of different concentrations of soybean lecithin (SL) and virgin coconut oil (VCO) in Tris-based extender on chilled and frozen-thawed bull semen quality parameters.
MATERIALS AND METHODS: A total of 24 ejaculates were collected from four bulls via an electroejaculator. Semen samples were diluted with 2% VCO in Tris-based extender which consists of various concentrations of SL (1, 1.25, 1.5, and 1.75%). A 20% egg yolk in Tris used as a positive control (C+). The diluted semen samples were divided into two fractions; one for chilling which were stored at 4°C for 24, 72, and 144 h before evaluated for semen quality parameters. The second fraction used for freezing was chilled for 3 h at 4°C, packed into 0.25 mL straws and then cryopreserved in liquid nitrogen. The samples were then evaluated after 7 and 14 days. Chilled and frozen semen samples were thawed at 37°C and assessed for general motility using computer-assisted semen analysis, viability, acrosome integrity and morphology (eosin-nigrosin stain), membrane integrity, and lipid peroxidation using thiobarbituric acid reaction test.
RESULTS: The results showed that all the quality parameters assessed were significantly (p<0.05) improved at 1.5% SL concentration in chilled semen. Treatment groups of 1, 1.25, 1.5, and 1.75% SL were higher in quality parameters than the control group (C+) in chilled semen. However, all the quality parameters in frozen-thawed semen were significantly higher in the C+ than the treated groups.
CONCLUSION: In conclusion, supplementation of 1.5% SL in 2% VCO Tris-based extender enhanced the chilled bull semen. However, there was no marked improvement in the frozen-thawed quality parameters after treatment.
Fulltext Butylated hydroxytoluene protects bull sperm surface protein-P25b in different extenders following cryopreservation

Khumran AM, Yimer N, Rosnina Y, Wahid H, Ariff MO, Homayoun H, et al.

Vet World, 2019 Apr;13(4):649-654.
PMID: 32546907 DOI: 10.14202/vetworld.2020.649-654

Aim: The aim of this study was to investigate the effects of different concentration of butylated hydroxytoluene (BHT) on sperm membrane surface protein "P25b" from cryopreserved bull semen in either lecithin based Bioxcell® (BX) or two egg-yolk based extenders, tris-egg yolk (TEY), and citrate-egg yolk (CEY).
Materials and Methods: Forty-five semen samples, 15 each were extended with either BX, TEY, or CEY extender which contained different concentrations (0.0 - control, 0.5, 1.0, 1.5, 2.0, and 3.0 mM/mL) of BHT. The extended semen samples were frozen at a concentration of 20×106/mL in 0.25 mL straws and stored in liquid nitrogen for 2weeks. The frozen samples were thereafter thawed, proteins extracted and analyzed for quantities of protein P25b through direct sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel densitometry. Peptides were confirmed by Western blotting (WB).
Results: Results showed that supplementation of BHT improved (p<0.05) quantity of protein P25b at concentrations of 0.5mM/mL for BX and at 1.0 mM/mL for TEY and CE when compared with the controls and other treatments.
Conclusion: BHT supplementation at 0.5 in BX and 1.0 mM/mL in TEY and CEY has protected bull sperm fertility marker protein P25b in frozen-thawed bull sperm.
Effect of different concentrations of egg yolk and virgin coconut oil in Tris-based extenders on chilled and frozen-thawed bull semen

Tarig AA, Wahid H, Rosnina Y, Yimer N, Goh YM, Baiee FH, et al.

Anim. Reprod. Sci., 2017 Jul;182:21-27.
PMID: 28511862 DOI: 10.1016/j.anireprosci.2017.03.024

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.

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