SEMINAL CHARACTERISTICS IN MURRAH BULLS RECOVERING FROM GOSSYPOL POISONING

The present research was undertaken to study the characteristics of fresh and cryo-preserved semen of elite pure breed Gir (Bos indicus) bulls. The mean values of fresh seminal parameters in neat semen viz. seminal volume (ml), sperm concentration (millions/ml), progressive sperm motility (%), live sperm (%), intact acrosome (%), total morphological sperm abnormalities (%), hypo osmotic swelling (HOS %) and sperm penetration distance (SPD- mm) were 4.99 ± 0.26, 895.33 ± 82.68, 69.10 ± 0.75, 72.16 ± 0.64, 84.42 ± 0.77, 15.96 ± 0.44, 60.12 ± 1.19 and 31.32 ± 0.70, respectively. Sperm concentration, individual motility, live sperm, total sperm abnormalities and sperm penetration distance differed significantly between bulls. The semen was extended, filled and sealed in 0.25 ml straws maintaining 20 million spermatozoa/straw and cryo-preserved using programmable bio freezer (IMV). Cryo-preserved semen was assessed 24 h after freezing and immediately after thawing. Freezing significantly lowered progressive sperm motility (69.10 ± 0.75 vs 53.81 ± 0.61), intact acrosome (84.42 ± 0.77 vs 75.69 ± 1.10), HOST (60.12 ± 1.19 vs 55.71 ±1.33) and CMPT (31.32 ± 0.70 vs 27.97 ±0.72). Whereas, significantly higher percentages of sperm abnormalities (15.96 ± 0.44 vs 16.92 ± 0.57) were observed after freezing.

The experiment was undertaken to study the efficacy of egg yolk free semen extender on cryopreservation of buffalo semen. A total of 24 semen ejaculates from Murrah bulls (5 to 6 years) maintained at Central Semen Station, Anjora, Durg, Chhattisgarh were divided into two aliquots and were diluted using Tris-fructose-egg yolk-glycerol (TFYG) and egg yolk free commercial extender (EYFCE, AndroMed). The overall average Murrah bull fresh semen volume (mL), sperm concentration (million/mL), initial progressive motility (%), live sperm (%), abnormal spermatozoa (%), intact acrosome (%) and HOS reactive sperm (%) were 4.90±0.49, 1286.92±100.23, 75.00±0.67, 81.45±0.61, 10.70±0.51, 80.16±0.43 and 68.83±0.40, respectively. There was significant difference between bulls in semen volume (P<0.01), individual motility (P<0.01) and total sperm abnormalities (P<0.05). The semen extended with TFYG and EYFC extenders were cryopreserved in french mini-straw (0.25 mL) using programmable bio-freezer. The mean values of post thaw motility (%), live sperm (%), abnormal sperm (%), intact acrosome (%), HOST (%) of Murrah bull semen extended with TFYG and EYFCE were 45.83±0.77 vs 49.16±1.33, 57.41±0.54 vs 58.62±0.73, 12.54±0.46 vs 12.45±0.63, 69.62±0.82 vs 71.45±0.64 and 56.45±0.52 vs 57.37±0.55, respectively. Post thaw motility, live spermatozoa and intact acrosome were significantly higher (P<0.01) in frozen thawed semen extended with as EYFCE compared to TFYG in Murrah bull semen. It is concluded that EYFCE results in improvement in post thaw semen characteristics viz. post thaw motility, live sperm percent and intact acrosome.

Melatonin improves testicular hemodynamics and sperm quality in rams subjected to mild testicular heat stress

Artificial insemination with frozen – thawed spermatozoa was introduced in most of the developing countries more than three decades ago, yet it has not been successfully applied in large scale. More than 50% spermatozoa are usually injured during the cryopreservation process. Injuries caused by cryopreservation are more likely due to increasing solute concentration and the formation of intracellular and extracellular ice crystals during cryopreservation [54] leading to significant decline in semen quality and alterations in sperm morphometrix. Therefore, the present study was designed to study cryopreservation of buffalo semen and the effect of semen cryopreservation on determinant like semen fertility viz. physico-morphologic. Forty eight semen samples collected from eight Murrah buffalo bulls maintained at Central semen station, Bhopal were included in the study for the study of physico-morphological characters of neat and cryopreserved semen. The overall average of the eight bulls for neat semen volume mass activity, progressive motility, live %, sperm concentration, head, midpiece, tail, and total sperm abnormalities was 2.89±0.14, 3.64±0.07, 71.56±0.37, 86.22±0.66, 963.92±38.51, 5.58±0.28, 2.18±0.32, 2.09±0.21 and 9.85±0.23 %, respectively. There was significant (P<0.05) between bulls and ejaculate variation in semen volume, sperm concentration, sperm midpiece and tail abnormalities. No significant differences between bulls as, however, recorded in sperm mass activity, progressive sperm motility, live sperm percent, sperm head and total sperm abnormalities.

Buffalo semen collected from Murrah bull were cryopreserved and evaluated for different motility parameter, kinematics and plasma membrane integrity. Buffalo bulls were maintained uniform standard management and nutritional practices. Semen was collected regularly twice a week semen collection schedule from four (04) Murrah bull. Collected semen was immediately transported to laboratory and evaluated for different macroscopic parameter (color, volume and thickness). Fresh semen was then diluted with saline solution and evaluated for sperm concentration, motility, sperm kinematics and morphology. Semen samples that fill all the standard were selected for freezing and diluted with Tris-egg yolk citrate diluter. Diluted semen was equilibrated, cryopreserved and finally evaluated for post thaw sperm quality. Different motility parameter (total, progressive, static and slow motility) varied significantly (p&lt;0.01) irrespective of different freezing stages. Significantly higher progressive sperm motility and viability of buffalo spermatozoa were observed at fresh semen whereas lower progressive sperm motility and viability was found at post thaw stage. Total and progressive motility reduced by 2.5 and 2.12% following equilibration, whereas following cryopreservation, total and progressive motility reduced by 35.7 and 28.51% and static motility increases accordingly (35.4%). Significantly higher plasma membrane integrity of sperm was observed at fresh semen followed by pre freeze and post thaw semen. Following freezing, integrity of plasma membrane reduces at the rate of 10.81% and 26.7% at pre freezing and post thaw stages. Significantly higher average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were found for fresh semen followed by pre-freeze and post-thaw semen. Frozen buffalo semen with higher progressive motility and motion characteristics may be produced if motility losses can be reduced during freezing stage as this stage results higher motility losses. Asian Australas. J. Biosci. Biotechnol. 2022, 7(2), 75-81

A study was carried out on semen ejaculates (40) of five healthy Surti buffalo bulls during favourable breeding season. The ejaculates with &gt;70% IM were diluted @ 100 million sperms/ml in Tris-fructoseegg yolk-glycerol (TFYG) diluent supplemented with L-cysteine @ 1 mg/ml, and examined for quality parameters such as sperm progressive motility, viability, morphology, acrosomal and plasma membrane integrity. A part of the extended semen was preserved at 5°C and another was processed for ultra-low temperature (-196°C) preservation in LN2 using biofreezer after filled in French mini straws. The mean values of ejaculate volume, sperm concentration, mass activity (0-5 score), individual sperm motility, live sperm, abnormal sperm, intact acrosome and HOS reactive sperms observed in fresh semen were 3.04±0.11 ml, 963.05±50.97 million/ml, 2.97±0.06, 75.00±0.69 %, 88.22 ±0.48 %, 4.40±0.26 %, 93.67±0.31 % and 89.17±0.57 %, respectively. Just after dilution the percentages of progressively motile, live &amp; abnormal sperms, intact acrosomes and HOS reactive sperms were 80.51±0.65, 85.95±0.65, 4.92±2.20, 91.00±2.30, 85.72±0.67, respectively. The corresponding values after 48 hrs of refrigeration (5°C) were 61.75±0.85, 67.85±0.85, 6.45±0.27, 89.05±0.32, 67.30±0.96%, the values at pre-freeze stage (after equilibration) were 72.62±0.69, 78.97±0.93, 5.45±0.25, 90.90±0.35, 78.12±0.79% and at post-thaw stage (37°C/30 sec) 46.50±0.72, 52.97±0.79, 7.10±0.26, 85.73±0.18, 51.62 ±0.82%, respectively. The mean motile spermatozoa observed after 1, 2 and 3 h of post-thaw incubation at 37°C in water bath were 32.75±0.82, 18.88 ±0.70 and 8.88±0.66% (P&lt;0.01), respectively. The semen quality parameters, fresh and cryopreserved were acceptable for artificial breeding use. The seminal traits in initial, 48 hrs refrigerated, pre-freeze and post-thawed samples revealed significant (p&lt;0.01) interrelationships (r = 0.44 to 0.84) between progressive motile sperm, live sperm and HOST reactive sperm directing more emphasis on these quality parameters for better semen evaluation.

The study was conducted to evaluate the seasonal influence (peak winter and summer) and the efficacy of three extenders (egg yolk based TFYG extender and egg yolk free soya bean based commercial extenders Optixcell and Andromed) on quality and freezability of Gir bull semen in Middle Gujarat. Semen ejaculates (6/bull/season, total36) revealed mean ejaculate volume 6.49±0.30 ml, sperm concentration1212.36±58.10 million/ml, progressive motility 74.17±0.78 %, live sperm 81.39±0.80 %, abnormal sperm 7.36±0.31 %, and sperm with intact plasma membrane 81.31±0.98 % and intact acrosome 94.81±0.24 %. Only the progressive sperm motility was significantly (P&lt;0.05) higher(76.39±0.97 % vs. 71.94±1.00 %) with lesser sperm abnormality(6.17±0.37 % vs. 8.56±0.30 %) during winter than in summer. Semen samples split diluted with TFYG, Optixcell and Andromed extenders recorded the overall mean values of progressive sperm motility, livability, abnormality, plasma membrane integrity and acrosomal integrity during winter season as 77.87±0.51, 77.50±0.45, 5.56±0.20,76.02±0.81 and 94.35±0.29 on dilution; 72.41±0.51, 70.50±0.64, 5.96±0.26, 71.20±0.79 and 93.09±0.32 at pre-freeze stage; 41.30 ±0.94, 50.28±1.03, 9.15±0.31, 29.89±0.40 and 90.65±0.40 at post-thaw stage, respectively. The respective values in summer season were 72.13±0.60, 75.50±0.60, 7.48±0.25, 75.61 ±0.55 and 94.09±0.30 on dilution; 65.46±0.66, 69.41±1.05, 8.89±0.28, 69.70±0.66 and 92.63 ±0.33 at pre-freeze stage; 31.48±0.52, 45.09±0.85, 13.48±0.33, 26.85±0.71 and91.26±0.38 at post-thaw stage. The overall mean sperm post-thaw motility/longevity at 0, 30, 60 and 120 min of incubation at 37°C was 41.20±1.51, 35.19±1.47, 28.80±1.75 and 17.50±1.47 % during winter season and 31.57±0.89, 26.20±0.77, 20.37±0.83 and 13.80±0.77% in summer season, respectively. The initial quality as well as freezability of semen in terms of motile, live, normal and HOS reactive sperm including post thaw longevity were better in winter season than in summer season. Further, the values of all the five semen quality parameters studied were comparatively better in Optixcell than TFYG and Andromed extenders with significant differences only in sperm progressive motility in both the seasons.The season x extender interaction was not significant for any of the sperm quality parameters studied.

Effect of kisspeptin injection on reproductive performance of Ossimi rams in subtropics

The aim of this work was to compare the effectiveness of ultra-rapid freezing (UF) and conventional slow freezing (CF) to cryopreserve buck sperm throughout the year. During 1 year, semen from 10 adult Gabon bucks was collected by electroejaculation every 2 weeks. Before and after freezing, samples were selected by density gradient centrifugation, and after sperm selection, the sample was divided into two aliquots. One aliquot was CF with an extender based on Tris, citric acid, and glucose (TCG) +6% yolk +5% glycerol, and maintained at 5°C for 3 hours of equilibration before freezing. The other aliquot was frozen using an UF method with an extender based on TCG +6% yolk +100 mM sucrose, and maintained at 5°C for 30 minutes. The evaluations included the percentages of motile sperm, sperm with progressive motility, quality of sperm motility, and the percentages of sperm with functional membrane, live sperm, sperm with morphoabnormalities, and sperm with intact acrosome. The percentage of sperm with intact acrosome was higher using the conventional freezing method (p < 0.05). After thawing and at pre- and postselection stages, the quality of motility, and the percentages of motile sperm, progressive motile sperm, sperm with functional membrane, and with intact acrosome were greater using CF than UF (p < 0.005). Conventional freezing was more effective than UF to cryopreserve sperm from Gabon bucks, at least in our experimental conditions. Most differences in favor of CF were observed in the quality of motility, and the percentages of motile sperm, progressive motile sperm, sperm with functional membrane, and with intact acrosome during long periods of the year, or even remained throughout it.

This experiment was conducted to study variations of serum testosterone and seminal characteristics of Markhoz male goats. Blood samples were obtained via jugular vein, and semen was collected by using an artificial vagina from 14 fertile male goats (2–3 years of age), at 15-day intervals starting on 15 July and ending on 30 October 2010 (during breeding and non-breeding season). Semen volume, total sperm (volume×concentration), live sperm (%), abnormal sperm (%) and semen pH were significantly superior during the late summer and early autumn (breeding season). Variation of sperm density, motility and progressive motility was not significant during the sampling period. The results presented show that the lowest and highest levels of lactate dehydrogenase in the seminal plasma were recorded in late October (2.82 U/ml) and in late August (4.81 U/ml), respectively. Moreover, the study indicated that the serum testosterone concentration was higher during late summer and early autumn (p<0.05) than at any other of sampling period. There were negative correlations between volume and sperm density (−0.135, p<0.05), and positive correlations between volume and percentage live sperm (0.224) and percentage progressive motility (0.194, p<0.01). Sperm density was correlated with live sperm (0.200, p<0.05) and progressive motility (0.202, p<0.01). The correlation between live sperm and progressive motility was 0.554 (p<0.01). Furthermore, the results in this study indicated a significant positive correlation between live sperm and LDH (0.450) and a negative correlation between sperm density and LDH concentration (−0.272) (p<0.01). Significant, but positive correlations were found between sperm motility and LDH (0.542) and testosterone concentration (0.522), respectively (p<0.05). In conclusion, this study demonstrated that the best obtained semen was collected in late summer (during decreasing photoperiod) and early autumn (September and October). This also coincides with the natural breeding season of Markhoz goats in Iran.

Creole cattle are an important zoogenetic resource that has been historically overlooked and marginalized. Consequently, their conservation is vital for preserving genetic biodiversity. The objective of this study was to evaluate the reproductive functionality of bulls from three Creole breeds through the seminal analysis of fresh, frozen, and post-thaw semen. Semen was collected using an artificial vagina from 60 bulls belonging to the Rodeo Creole, Longhorn, and Senepol breeds. The evaluated characteristics in fresh semen included body weight (BW), scrotal circumference (SCi), ejaculate volume (EV), sperm concentration (SCo), mass sperm motility (MSM), individual sperm motility (ISM), pH, viability, and total abnormal sperm (TA). In addition, semen straws were frozen, and the post-thaw individual sperm motility (PISM) was evaluated. Senepol bulls exhibited higher BW, SCi, and PISM, as well as lower TA (567.3 kg, 35.95 cm, 64.90% and 8.8%, respectively) compared to Rodeo Creole and Longhorn bulls. SCo and sperm viability were similar between Senepol and Longhorn bulls (P 0.05). No significant differences were found among the three Creole breeds for EV and MSM. BW and pH were significantly correlated (P 0.05) with sperm viability and TA percentage; SCi was correlated with EV; MSM with ISM, viability, pH and TA; and ISM was correlated with viability, TA and PISM. Additionally, sperm viability was correlated with both TA and PISM, and TA was also correlated with PISM. In conclusion, the quality parameters of fresh semen in these Creole bulls fall within the established reference values for the bovine species. Therefore, their semen can be cryopreserved for conservation and genetic dissemination purposes.

The study was undertaken to know the characteristics of various attributes of ejaculates collected from 6 elite pure Tharparkar bulls using artificial vagina in fresh and cryopreserved semen. The mean values for fresh seminal parameters, viz. seminal volume (ml), mass motility (0–5 scale), sperm concentration (millions/ml), individual motility (%), live sperm (%), intact acrosome (%), total sperm abnormalities (%), hypo osmotic swelling test (HOST -%) and cervical mucus penetration test (CMPT – mm) were 4.40 ± 0.24, 3.15 ± 0.12, 995.16 ± 70.99, 71.88 ± 0.99, 67.23± 1.08, 79.65 ± 1.21, 19.87± 0.54, 60.15 ± 1.76 and 31.56 ± 0.96, respectively. Semen volume and sperm concentration significantly differed between bulls within breed. The semen was cryopreserved after extension maintaining 20 million sperm/ straw, filling and sealing in 0.25 ml straw using programmable bio- freezer (IMV). Cryopreserved semen was assessed after 24 h freezing after thawing. Freezing significantly lowered motility (71.88 ± 0.99 vs 51.16 ± 1.08), intact acrosome (79.65 ± 1.21 vs 72.43 ± 1.42), HOST (60.15 ±1.76 vs 55.81 ±1.72) and CMPT (31.56 ± 0.96 vs 26.90 ± 0.91). Significantly higher percentages of sperm abnormalities (19.87 ± 0.54 vs 23.50 ± 0.82) were observed after freezing. Freezing resulted in significant decline in post thaw motility, acrosomal integrity, HOST and CMPT and increase in sperm abnormalities in Tharparkar bull semen.

Flow cytometric assessment of fresh and frozen-thawed Canada goose (Branta canadensis) semen

In South American camelids, raw semen only presents sperm with oscillatory movements. Therefore, it is necessary to treat these cells to enable them to acquire progressive motility. The effects of raw seminal plasma (SP) on sperm movement patterns (oscillatory, progressive, and hyperactive) have apparently not yet been reported. The objective of this study was to determine effects of raw seminal plasma on sperm motility, viability, and acrosomal status in fresh llama semen. A total of 15 ejaculates were collected (electroejaculation) from 5 llamas (n = 5, r = 3). Each ejaculate was diluted 4 : 1 in 0.1% collagenase in HEPES-TALP (HT) medium and incubated 4 min at 37°C, with the objective of separating spermatozoa from SP. Immediately after incubation, each ejaculate was divided into 2 and centrifuged for 8 min at 800 × g. Pellets were resuspended in either HT or raw SP and maintained at 37°C until evaluation (at 0, 1.5, and 3 h). Sperm motility was evaluated using a phase contrast microscope and a warm stage. Propidium iodide and carboxyfluorescein diacetate were used for assessing membrane integrity (viability). Acrosomal status was evaluated with the Coomassie blue stain. A split-plot design was used with treatment as a factor, with 2 levels (HT and SP) and time as the other factor, with 3 levels (0, 1.5, and 3 h), and blocked by males. There was no significant interaction between treatments (HT and SP) and times (0, 1.5, and 3 h) for each of the seminal characteristics evaluated. Progressive sperm motility was observed after collagenase treatment in all samples. Progressive motility disappeared immediately after the addition of raw SP and showed only oscillatory movements. In contrast, samples incubated in HT maintained progressive motility and became hyperactive. There were no differences (P &gt; 0.05) in total motility of sperm incubated in HT among incubation times (0 h: 30.8 ± 18.9%; 1.5 h: 26.5 ± 11.5%; and 3 h: 21.5 ± 13.5%). However, in samples incubated with SP, a decrease (P &lt; 0.05) in total sperm motility was detected after 3 h of incubation (0 h: 16.5 ± 12.6%, 3 h: 2.3 ± 3.2%). Sperm viability was not different (P &gt; 0.05) between treatments (HT and SP); samples incubated in HT retained 78.4% of the initial viability (32.8/41.8, 3 h/0 h), and samples incubated in SP retained 69.7% of their initial viability (24.4/35.0, 3 h/0 h). The percentage of spermatozoa with intact acrosomes was not different (P &gt; 0.05) between treatments (HT and SP); however, the percentage of sperm with intact acrosomes decreased after 3 h of incubation in both samples (HT and SP). Due to the presence of a high percentage of progressive and hyperactive motile sperm in samples incubated in HT and their absence in samples incubated in SP, we concluded that raw seminal plasma preserved oscillatory sperm motility. Further studies are needed to understand the effects of SP on South American camelid spermatozoa.

A study was carried out to evaluate the effect of different equilibration periods on the postthaw seminal traits of buck semen collected at weekly intervals from the adult Jamunapari bucks (2–3 years old), maintained under intensive system of management at Central Institute for Research on Goats, Makhdoom. A total of 17 ejaculates were processed for freezing having the volume ≥ 1.0 ml and mass activity of +4.0 and above. The ejaculates were processed for conventional freezing in two step dilution to maintain the final dilution rate of 1: 10 having 6% glycerol and 10% egg yolk levels. The samples were kept for 4 h in cold handling cabinet and at each hour of equilibration period (1, 2, 3, 4 h), samples were packaged in 0.25 ml French mini straws and frozen in horizontal vapour freezing. Pre-freeze/equilibration progressive motility, live and total abnormal sperm count were done at the end of each h of equilibration period. Then samples were thawed at 40°C for 45 sec after 24 h of storage and the post-thaw progressive motility, live and total abnormal sperm count were carried out. The pre-freeze and post-thaw progressive motility at 1, 2, 3 and 4 h of equilibration periods were 83.24±1.05 and 30.59±2.42, 81.76±0.85 and 31.47±1.76, 74.71±1.39 and 33.24±2.10, 70.59±1.28 and 28.82±1.99%, respectively. The respective values for live sperm count were 88.44±1.21 and 46.02±2.83, 80.85±5.36 and 41.95±1.56, 81.90±2.06 and 43.57±3.53, 80.37±2.32 and 41.01±3.02%. The total abnormal sperm ranged from 0.91 to1.74% before freezing and 2.18 to 2.84% after freezing at different hours of equilibration periods. The pre-freeze progressive motility up to 2 h of equilibration period varied significantly (P<0.05) with 3 and 4 h of equilibration periods. The remaining characteristics did not show any significant variation between different hours of equilibration periods. Therefore, equilibration period of 3 h may be followed in the freezing protocol used for buck semen cryopreservation as equilibration period of 2 or 3 h showed superiority over 1 or 4 h in terms of post-thaw spermatozoan characteristics in frozen-thawed Jamunapari buck semen.