• Animal Bioscience
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• v.34 no.2
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• pp.192-197
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• 2021

Objective: The present study evaluated the preservation of ram semen at 0℃ using soybean lecithin with a Tris-fructose extender. Methods: Semen was collected by artificial vagina ejaculation from six rams with proven fertility. High quality ejaculates were diluted by soybean lecithin (0.25%, 0.5%, 0.75%, 1.0%, 1.25%) using Tris-fructose extender and control (Tris-fructose egg yolk extender), respectively. The ejaculates were diluted to a concentration of 5×108 sperm/mL, followed by cooling to 0℃ in 90 min and maintaining the temperature for 12 days. The diluted semen samples were examined and recorded for sperm progressive motility, acrosome integrity at 0, 24, 72, 144, 216, 288 h, respectively. Two hundred and twenty-three ewes were inseminated for 216 h with optimal soybean lecithin concentrated semen or control via trans-cervical insemination. Results: The results showed that there were no differences in sperm progressive motility at 0, 24, 72, and 144 h (p>0.05). After 216 h, the sperm progressive motility in the control group and 0.5% concentration groups was significantly higher when compared to 0.25% concentration (p<0.05). The 0.5% concentration group demonstrated the highest survival rate and had no difference with the control group (p>0.05). At 216 h, the sperm progressive motility of all groups was still above 50%. The acrosome integrity of all groups was decreased with prolongation of storage time, but there was no difference at each time point (p>0.05). There was no significant difference in the lambing rate and pregnancy rate between the 0.5% concentration group and the control group (p>0.05). Conclusion: These results suggest that ram sperm is capable of fertilization after preservation at 0℃ with 0.5% of soybean lecithin in Tris-based extender substituted for egg yolk and produce normal offspring after insemination.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.11
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• pp.1527-1533
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• 2001

Garole is a prolific, rare, less known and small size Indian sheep breed found in low and humid Sunderban region of West Bengal. Although information on stored Garole ram liquid semen upto 24 h is available, but there is a need to further investigate the short-term and long-term preservability of Garole ram semen for extensive utilization of this valuable germplasm by artificial insemination. The aim of the present study was to apply computer-assisted sperm analysis technique for assessing the motion characteristics of Garole ram semen stored (i) in liquid state at refrigeration temperature for short-term preservation upto 48 h and (ii) in frozen state at $-196^{\circ}C$ for long-term preservation after packaging in mini straws. Short-term preservation had a significant effect on motility (p<0.01) as the motility progressively decreased from 90.1% at 0 h to 85.5% and 73.2% after 24 and 48 h of storage, respectively. Although the decline in rapid moving sperms was also significant (p<0.01) on storage but the decrease was more pronounced at 48 h as compared to 24 h of storage period. Storage of chilled semen had also a significant effect on % linearity (p<0.05), % straightness (p<0.01), sperm velocities (p<0.01), amplitude of lateral head displacement (p<0.01) and beat frequency (pO.Ol) of spermatozoa. The replication had a significant effect for all the variables except average path and straight line velocity. However, the interactions of short-term storage and replication were non-significant for most of the variables except % of medium moving sperms, sperm velocities and beat frequency. On long-term preservation of Garole ram spermatozoa under controlled conditions the mean post-thaw recovery of 70.4 and 71.4% motile spermatozoa was achieved having 48.8 and 48.9% of rapidly motile spermatozoa, respectively in both the replicates. The effect of replication on cryopreservation was significant (p<0.05) on amplitude of lateral head displacement and beat frequency, but there was no significant effect on motility, rapidly motile spermatozoa, linearity, straightness and sperm velocities of frozen-thawed spermatozoa. It can be concluded from these results that an average 70% motility can be achieved on storage of Garole ram semen in chilled liquid state upto 48 h or in liquid nitrogen after freezing under controlled conditions in straws. However, further studies are required to evaluate the fertility of short-term and long-term preserved Garole ram semen for extensive use of this prolific sheep breed.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.4
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• pp.212-219
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• 2021

The aims of this study were to measure the ultrasonographic biometry of genitalia of the indigenous rams and observe the relationship of biometry on semen parameters. The epididymal volume was significantly reduced (p < 0.01) after semen collection compared with before collection for both left and right part in all rams. The cumulative results showed that although there was no significant difference in length, width and volume of epididymis between before and after semen collection, however the values were lower after collection. The epididymal length was significantly correlated with epididymal volume (p < 0.01), semen motility (p < 0.05) and semen morphology (p < 0.01). Epididymal width was only significantly correlated with epididymal volume (p < 0.01) not with the semen parameters. Epididymal volume had a significant correlation only with semen morphology (p < 0.01).The scrotal circumference had the significant correlation with semen density, mass activity, concentration and motility (p < 0.01). The epididymis had the similar or slightly increased echogenicity as compared to the normal testis. During whole study, some white spots were found on testis which did not affect the semen quantity and quality. Significant variation was observed only for semen concentration and motility among the rams (p < 0.05). The overall normal morphology was 90.5 ± 4.6% with highest percentage of coiled tail abnormalities.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.10
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• pp.1276-1281
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• 2010

The present study was conducted to investigate the effects of butylated hydroxytoluene (BHT) supplementation on diluted, cooled and frozen-thawed ram spermatozoa. After primary evaluation of collected ejaculates, only semen samples with motility of more than 70% and sperm concentration higher than $3{\times}10^3$ sperm/ml were used for cryopreservation. The selected semen samples were then pooled and diluted 1:4 with Tris Citrate Fructose Yolk (TCFY) extender supplemented with different concentrations of BHT (0.5, 10, 2.0 and 3.0 mM). As the control, semen was diluted and frozen in the diluent without BHT. Motility, progressive motility, viability, membranes and acrosome integrity were evaluated after dilution (part 1), cooling (part 2) and freezing and thawing (part 3). The results of the first part of the experiment showed that there were no significant difference between treatments in the motility, progressive motility, viability, membranes and acrosome integrity of spermatozoa, but the results with 2.0 mM BHT were slightly better than obtained with other levels of BHT and control extender. Significantly better results (p<0.05) were observed in the second part of the experiment for cooled spermatozoa characteristics, when extender was supplemented with 2.0 and 3.0 mM BHT. Furthermore, the results obtained in the third part of the experiment indicated that, after freezing and thawing, all evaluated semen characteristics were improved significantly (p<0.05) by increasing BHT levels, with the best results obtained for extender containing 2 mM BHT. Comparison of these results with those of control diluent, the effects of supplementation were significantly (p<0.01) better. However, the higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of spermatozoa compared to extender containing 2.0 mM BHT. In conclusion, the results obtained in this study showed that the semen quality of rams was improved when BHT was added to extender used before the freezing process.

• Journal of Embryo Transfer
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• v.28 no.4
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• pp.325-335
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• 2013

The study was set for one year to measure the effects of concentrate supplementation on reproductive performances and semen quality in indigenous rams. The study was conducted at the Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh during the period from May 2011 to April 2012. Forteen ram lambs (4~5 months) were randomly divided into two equal groups (n=7); supplemented vs control. The animals of control group were maintained on natural grazing. Along with natural grazing the supplemented group was on supplemented feeding. The concentrate supplementation (Wheat bran, Crushed maize, Soy bean meal, Fish meal, DCP powder, Vitamin mineral premix, Salt) was provided @ 300 g/head /day to the supplemented group. Body weight, scrotal circumference, BCS and libido index were measured weekly. Age, body weight and scrotal circumference at puberty were recorded. Semen was collected once in a weak using artificial vagina and chilled at $5^{\circ}C$ for 48h for evaluation. Concentrate supplementation did not influence (p>0.05) body condition score, age, weight, scrotal circumference at puberty and libido index. Final body weight (kg), growth rate (g/d), scrotal circumference (cm) and scrotal growth rate (mm/15d) were significantly (p<0.05) higher in supplemented group of rams compared to control. Volume, concentration, motility and membrane potentiality of spermatozoa were varied significantly (p<0.05) in supplemented and control groups. However, density, mass motility, viability and sperm with normal acrosome, midpiece and tail were not differed insignificantly (p>0.05) in different observation times. It was concluded that concentrate supplementation with free grazing improved weight and scrotal circumference gain and semen production with increased quality in indigenous ram.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.3
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• pp.318-325
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• 2010

Two experiments were designed to evaluate the effects of the amino acids glycine and cysteine on cryopreservation of ram spermatozoa. After primary evaluation of collected ejaculates, the semen samples were pooled and diluted 1:4 before cooling (experiment 1) and freezing (experiment 2) with Tris-Citrate-Fructose-Yolk (TCFY) extender supplemented with different concentrations of glycine and cysteine (5, 10, 15 and 20 mM). As the control, semen was diluted and frozen in the extender without amino acids. Motility, viability and membrane integrity were assessed as the parameters for semen quality in the first experiment. In the second experiment, motility, progressive motility, viability, membranes and acrosome integrity were evaluated after the freezing-thawing process. The results of the first experiment indicated that the addition of 10 and 15 mM cysteine compared to the control (basic) extender significantly increased (p<0.01) the motility, viability and membrane integrity of spermatozoa after cooling. However, further increasing these amino acids up to 20 mM had a significant negative effect (p<0.05). Our results showed no significant differences (p>0.05) between 5 mM glycine compared to 5 mM cysteine and between 20 mM glycine and 20 mM cysteine. The results of experiment 2 showed that the amino acids significantly improved post-thaw motility, progressive motility, viability, membranes and acrosome integrity of ram spermatozoa. These positive effects were observed at concentrations between 5 to 15 mM of glycine and cysteine, with the best results at 15 mM. Further increasing of amino acid concentrations significantly decreased the post-thaw characteristics of spermatozoa, but the results showed that cysteine was better than glycine and control extenders. The data indicated that addition of glycine or cysteine to the freezing extender can be recommended for cryopreservation of ram spermatozoa. However, further studies are still needed to determine the effect of such addition on fertility in farm animals.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.12
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• pp.1716-1721
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• 2006

The present study was conducted to observe the effect of osmolality of glycerolated TEST-yolk glycerol extenders on post-thawing sperm kinematics of ram spermatozoa of the native Malpura breed maintained in a semi-arid tropical environment. Good quality semen obtained from adult rams was pooled, split and diluted to 1,000 million spermatozoa per ml in complete TEST-yolk-glycerol extenders of 900, 1,200, 1,500 and 1,800 mOsm/kg osmolality. Diluted semen samples were loaded in 0.25 ml straws and cooled down to $-125^{\circ}C$ freezing temperature at the rate of $-25^{\circ}C$ per minute under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at $50^{\circ}C$ in a water bath for 10 seconds and sperm kinematics of the frozen-thawed spermatozoa were assessed by a computer-assisted sperm analysis technique. Osmolality of diluent had no significant effect on post-thawing % motility, % rapid, % medium and % slow moving frozen-thawed spermatozoa but significantly (p< 0.05) affected the % linearity and % straightness. The post-thawing % motility and % rapid motile spermatozoa were highest in samples extended in diluent of 1,500 mOsm/kg osmolality and lowest in 900 mOsm/kg. The curvilinear velocity of spermatozoa was significantly (p<0.05) higher for samples extended in 1,800 mOsm/kg, compared to those in 900 and 1,200 mOsm/kg, but the effect was not significantly different to those extended in diluent of 1,500 mOsm/kg osmolality. The study indicated that ram spermatozoa could tolerate a wide osmolality range for dilution in the complete TEST-yolk-glycerol extender for their cryosurvival. The highest recovery of motile spermatozoa following thawing was achieved in samples extended in the TEST-yolk-glycerol diluent of 1,500 mOsm/kg osmolality.

• Animal Bioscience
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• v.37 no.5
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• pp.852-861
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• 2024

Objective: The present study aimed to investigate the effect of β-nicotinamide mononucleotide (NMN) supplementation on ram sperm quality during storage at 4℃ in vitro. Methods: Tris-citric acid-glucose solution containing different doses of NMN (0, 30, 60, 90, and 120 µM) was used to dilute semen collected from rams and it was stored at 4℃. Sperm motility, plasma membrane integrity as well as acrosome integrity were evaluated at 0, 24, and 48 h time points after storage at 4℃. In addition, sperm mitochondrial activity, lipid peroxidation (LPO), malondialdehyde (MDA) content, reactive oxygen species (ROS) content, glutathione (GSH) content, superoxide dismutase (SOD) activity, and apoptosis were measured at 48 h time point after storage at 4℃. Results: Results demonstrate that the values obtained for sperm motility, acrosome integrity, and plasma membrane integrity in the NMN treatments were significantly higher than control (p<0.05). The addition of 60 µM NMN significantly improved ram sperm mitochondrial activity and reduced LPO, MDA content, and ROS content compared to control (p<0.05). Interestingly, sperm GSH content and SOD activity for the 60 µM NMN treatment were much higher than those observed for control. NMN treatment also decreased the level of Cleaved-Caspase 3, Cleaved-Caspase 9, and Bax while increasing Bcl-2 level in sperm at 48 h time point after storage at 4℃. Conclusion: Ram sperm quality can be maintained during storage at 4℃ with the addition of NMN at 60 µM to the semen extender. NMN also reduces oxidative stress and apoptosis. Overall, these findings suggest that NMN is efficient in improving the viability of ram sperm during storage at 4℃ in vitro.

• Journal of Animal Science and Technology
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• v.56 no.3
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• pp.8.1-8.6
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• 2014

Background: Artificial insemination (AI) can serve as a powerful tool to the sheep owners for making rapid genetic progress of their flock. The AI in sheep is mostly performed using fresh semen with two reasons i) lambing rate following trans-cervical AI with frozen semen is limited by the inability of frozen-thawed sperm to transit the cervix and ii) the need of circumventing the cervical barrier through laparoscope aided intrauterine AI. Therefore, AI with frozen-thawed semen is not as widespread in sheep as it is in other domestic species. However, to get maximum benefits through the use of AI, frozen-thawed semen is a prerequisite because instead of high fertility, the short shelf life of fresh semen coupled with a limitation on the number of insemination doses achievable per unit time restricts the widespread use of individual sires. Therefore, in order to enhance lambing rate, a total of 240 trans-cervical artificial inseminations with frozen-thawed semen were performed in Bharat Merino ewes during autumn season either once in the evening (G-I, 10 h after onset of estrus, n = 100) or twice (G-II, 14 h and 22 h after onset of estrus, n = 140) i.e. once in the morning and again in the evening. Results: The pregnancy rate (proportion of pregnant ewes confirmed by ultrasonography at day 40) and lambing rate (proportion of ewes lambed) were higher in G-II as compared to G-I (26.4 vs 20% and 19.3 vs 10%, respectively). The difference in lambing rates was statistically (P < 0.05) significant. The depth of insemination within cervico-uterine tract had no significant effect on pregnancy and lambing rates. Conclusions: The results indicate that lambing rate in sheep following TCAI with frozen-thawed semen was significantly influenced by time of inseminations. Two inseminations after 14 and 22 h of onset of estrus enhanced the lambing rates of Bharat Merino sheep as compare to single insemination after 10 h of onset of estrus. The TCAI technique with frozen-thawed ram semen is promising and may serve as a valuable tool for genetic improvement of sheep breeds. Research efforts are going on worldwide to overcome the poor fertility following TCAI with frozen-thawed semen.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.5
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• pp.633-636
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• 2002

Six adult Garole rams maintained under a semi-intensive system were used as semen donors for this study. Semen was collectied daily during the monsoon season with the help of an artificial vagina and examined for its quality characteristics. Ejaculates of thick consistency, rapid wave motion, ${\geq}80%$ motility and intense movement of motile spermatozoa were diluted at the rate of 1:1 with egg yolk McIllvaine glucose diluent at $30^{\circ}C$ in water bath. Estrus in ewes was detected by parading aproned rams of proven vigour at 12 h intervals. The ewes (54 Malpura and 23 Avikalin) in estrus were artificially inseminated with fresh diluted ram semen. The overall conception rate was 94.8%, (range 91.7 to 100%). The overall lambing percent was 80.5 with a range of 75.0 to 84.6%. There was no significant (p>0.05) difference in lambing and conception rate because of individual rams. Fertility was significantly lower (p<0.05) in ewes of less than two years and more than six years of age. Breed (Malpura and Avikalin) effect was not observed in conception and lambing rate (p>0.05). No significant difference (p>0.05) in birth weight and 12 month weight was observed between Garole${\times}$Avikalin and Garole${\times}$Malpura crossbred lambs but there was significant (p<0.05) difference at three month and six months body weight of both the crossbred lambs.