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Summary: The free radical scavenging activity (RSA) of Nigella sativa extract and its efficiency for cryopreservation of buffalo spermatozoa was investigated. In experiment 1, Nigella sativa extract was prepared and evaluated for RSA using 2,2‐diphenyl‐1‐picrylhydrazyl assay. The results showed increased pattern of RSA at 1%–5% of Nigella sativa extract. In experiment 2, buffalo semen from three bulls (24 ejaculates) was incubated at 0%, 0.1%, 0.3%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5% and 6% extract to assess in vitro tolerability to Nigella sativa in terms of progressive motility (PM). Buffalo spermatozoa showed tolerance to all levels; rather, sperm PM was increased at 1%–4% extract. In experiment 3, semen from three bulls (24 ejaculates) was cryopreserved with 0%, 1%, 2%, 3%, 4% and 5% of Nigella sativa extract. Sperm PM and plasma membrane integrity (PMI) were evaluated after dilution and cooling, while PM, PMI, viability and DNA integrity were evaluated after thawing. Nigella sativa extract at 4% in extender improved (p < .05) post‐dilution, post‐cooling and post‐thaw sperm quality. In conclusion, Nigella sativa extract at all concentrations (1%–6%) showed antioxidant activity and its supplementation at 4% in extender improved buffalo sperm quality at all stages of cryopreservation. [ABSTRACT FROM AUTHOR]

Contents The study was designed to evaluate AndroMed® for the freezability and fertility of Nili-Ravi buffalo semen. Semen was collected from four adult Nili-Ravi buffalo ( Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 106 spermatozoa/ml) in tris-citric egg yolk or AndroMed® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post-thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar ( p > .05) in tris-citric egg yolk and AndroMed® extender. Higher ( p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed® compared to tris-citric egg yolk post-thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower ( p < .05) in frozen-thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ ( p > .05) either cryopreserved in tris-citric egg yolk or AndroMed® extender (45.5% vs. 49%). It is concluded that AndroMed® is capable in protecting the buffalo bull sperm during freeze-thawing process and can be adopted safely for routine use replacing the tris-citric egg yolk extender in artificial insemination programme. [ABSTRACT FROM AUTHOR]

This study was designed to evaluate the effect of thioglycol in extender on post-thaw quality of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen from three buffalo bulls was collected at weekly intervals (three replicates; two ejaculates per bull per replicate) and qualifying ejaculates (n=18) were cryopreserved in extenders containing thioglycol at 0.0 or 0.5 or 1.0mM. Sperm motility (%; visually), plasma membrane integrity, (%;supravital hypo-osmotic swelling test), viability (%;dual staining technique using Trypan blue and Giemsa stains) and DNA integrity (%;acridine orange test) was assessed at 0, 2 and 4 hour post-thaw. Sperm motility, plasma membrane integrity and viability of buffalo bull spermatozoa were improved in a dose dependent manner with the supplementation of thioglycol at 0.5 mM and 1.0mM compared control. Sperm DNA integrity was similar (P>0.05) in extenders containing 0.5 mM and 1 mM thioglycol that remained higher (P < 0.05) than the values of DNA integrity in control. It is concluded that 1.0mM thioglycol in extender improved the motility, plasma membrane integrity, viability and DNA integrity of buffalo bull spermatozoa. [ABSTRACT FROM AUTHOR]

Abstract: This study was designed to determine whether low-density lipoporoteins (LDLs) extracted from egg yolk in extender improve the freezability and fertility of buffalo bull semen. Semen from three Nili-Ravi buffalo bulls was diluted at 37 °C with tris-citric acid extender (50 × 106 motile spermatozoa mL−1) containing LDLs 2.5%, 5%, 10%, and 15% extracted from egg yolk and extender containing 20% egg yolk was kept as control. Diluted semen was cooled to 4 °C in 2 h, equilibrated at 4 °C for 4 h, filled in 0.5 mL French straws, and kept on liquid nitrogen vapors for 10 min. Straws were then plunged and stored in liquid nitrogen (–196 °C). Sperm motility (visually; %), plasma membrane integrity (%; with supravital hypo-osmotic swelling test), and viability (%; with dual staining test using Trypan-blue Giemsa) were assessed at post-dilution, pre-freezing and post-thawing. At post-dilution and pre-freezing, sperm progressive motility, plasma membrane integrity and viability was similar (P > 0.05) in extender containing 10% LDLs or the control. However, at post-thaw the aforementioned parameters were higher (P < 0.05) in extender containing 10% LDLs compared with the control and other experimental extenders. The fertility rate of inseminations performed were higher (P < 0.05) with extender containing 10% LDLs than the control. It was concluded that LDLs (10%) in extender improved the freezability and fertility of buffalo bull spermatozoa. [Copyright &y& Elsevier]

Abstract: This study was designed to compare commercially available extender Bioxcell® with tris-citric egg yolk extender for post thaw quality and in vivo fertility of buffalo semen. For comparison of post thaw semen quality: semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina (at 42 °C) for three weeks (replicates). Qualifying ejaculates having motility >60% from each buffalo bull were divided in two aliquots and diluted (at 37 °C having 50 × 106 spermatozoa/ml) in tris-citric egg yolk or Bioxcell® extender. Diluted semen was cooled to 4 °C in 2 hours, equilibrated for 4 hours and filled in 0.5 ml straws. Semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. Straws were then plunged and stored in liquid nitrogen (−196 °C). After 24 hours of storage, semen straws were thawed at 37 °C for 30 seconds to assess sperm motility, viability, plasma membrane integrity, normal apical ridge, and abnormalities (head, mid piece, and tail). For comparison of in vivo fertility: semen from two buffalo bulls of known fertility was cryopreserved in tris-citric egg yolk and Bioxcell® as described earlier, and used for inseminations under field conditions. Post-thaw percentage of sperm motility (45.3 ± 1.1, 45.0 ± 1.4), viability (66.2 ± 1.1, 64.4 ± 1.3) plasma membrane integrity (60.4 ± 1.2, 59.2 ± 1.4) and normal apical ridge (82.9 ± 0.5, 80.7 ± 0.5) did not differ (P > 0.05) in tris-citric egg yolk and Bioxcell® extender, respectively. Similarly, sperm abnormalities of head (1.20 ± 0.1, 1.20 ± 0.1), mid piece (0.67 ± 0.1, 0.87 ± 0.1) and tail (11.7 ± 0.2, 11.6 ± 0.3) remained similar (P > 0.05) in tris-citric egg yolk and Bioxcell® extender, respectively. In vivo fertility rates of buffalo semen cryopreserved in tris-citric egg yolk and Bioxcell® also remained similar (44% vs. 47%). It is concluded that commercially available Bioxcell® may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg yolk extender. [Copyright &y& Elsevier]

The population of red jungle fowl is declining and needs special attention for its conservation with suitable approaches. For ex situ in vitro conservation of Indian red jungle fowl, establishment of semen cryobank is an appropriate option, for which an extender with adequate retrieval capacity for functional spermatozoa is required. Therefore, studies were designed to evaluate a wide range of extenders for cryopreservation of Indian red jungle fowl ( Gallus gallus murghi ) sperm to achieve maximal post-thawed semen quality and fertility. For this purpose, semen from eight mature cocks were collected, initially evaluated (percent sperm motility, volume and concentration), pooled, assessed for motility, plasma membrane integrity, viability and acrosome integrity, and divided into six aliquots for dilution (1:5; 37 °C) in Beltsville poultry, red fowl extender, Lake, EK, Tselutin poultry and chicken semen extenders. Diluted semen was cooled from 37 °C to 4 °C @ −0.275 °C/min. Glycerol (20%) was added to chilled semen, equilibrated for 10 min, filled in 0.5 mL French straws, kept over LN 2 vapours for 10 min and plunged into LN 2 and stored at −196 °C. Percentages of motility, plasma membrane integrity, viability and acrosome integrity were higher (P < 0.05) in red fowl extender at 0, 2 and 4 h of incubation post-thaw. After cryopreservation and post-thawing at 37 °C the highest (P < 0.05) recovery rates and absolute livability index was also recorded in red fowl extender that was thus used for further artificial insemination of cooled-diluted (Liquid) and cryopreserved sperm. The no. of fertilized eggs (Liquid, 20.6 ± 0.4; Cryopreserved, 12.6 ± 0.5), percent fertility (86.7 ± 2.2; 57.2 ± 3.9), no. of hatched chicks (18.2 ± 0.8; 10.0 ± 0.3), percent hatch (76.5 ± 2.7; 45.3 ± 2.2) and hatchability of fertilized eggs (88.3 ± 3.4; 79.6 ± 3.4) were higher with sperm respectively freshly cooled-diluted or cryopreserved in red fowl extender. However, the rates obtained with frozen-thawed sperm were already successful for cryo-banking purpose and artificial insemination practice. In conclusion, we show the first fertility success obtained with cryopreserved Indian jungle fowl sperm. In addition, the red fowl extender is superior in maintaining the quality of Indian red jungle fowl cryopreserved sperm compared to Beltsville poultry, Lake, EK, Tselutin poultry and chicken semen extender. [ABSTRACT FROM AUTHOR]

The present study was conducted to compare the liposome-containing, animal protein–free, commercially available OPTIXcell extender with the Tris –citric–egg yolk extender for postthaw quality and fertility of buffalo semen. Semen was collected from five adult Nili-Ravi buffalo ( Bubalus bubalis ) bulls of similar age group with an artificial vagina (at 42 °C) for 3 weeks (replicates). Semen ejaculates from each buffalo bull were divided into two aliquots and diluted (at 37 °C having 50 × 10 6 spermatozoa/mL) in the OPTIXcell or Tris –citric–egg yolk (control) extender. Diluted semen was cooled to 4 °C in 2 hours, equilibrated for 4 hours, and filled in 0.5-mL straws. The semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. The straws were then plunged and stored in liquid nitrogen (−196 °C). After 24 hours of storage, the semen straws were thawed at 37 °C for 30 seconds to assess postthaw quality. Percentages of sperm motility, plasma membrane integrity, viability, and acrosomal integrity were improved (P < 0.05) in the OPTIXcell extender compared to the Tris –citric–egg yolk extender. Values for DNA integrity (%) did not differ in the OPTIXcell and Tris –citric–egg yolk extenders. The overall conception rate in buffaloes was improved (P < 0.05) with semen cryopreserved in the OPTIXcell extender (59.5%) compared to semen cryopreserved in the Tris –citric–egg yolk extender (41.5%). It is concluded that the liposome-containing commercially available OPTIXcell extender is more efficient to conserve postthaw quality and resulted in higher fertility rate of buffalo in the field. [ABSTRACT FROM AUTHOR]

Alpha linolenic acid (ALA) is integral component of cell membrane that protects the cell in stressful events and involves in many metabolic pathways. It was hypothesized that ALA have the ability to protect the structural and functional integrity of buffalo spermatozoa during freeze-thawing. Therefore, study was designed to evaluate ALA supplementation (0, 5, 10 and 20 ng/mL) in extender on freezability and in vivo fertility of buffalo bull spermatozoa. Semen from three adult Nili-Ravi buffalo bulls of similar age was collected with artificial vagina (42 °C) for five weeks (replicates; N = 30). Qualified semen ejaculates (>1 mL volume, >60% motility; >0.5 billion/mL concentration) were diluted with tris -citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/mL ALA at 37 °C and cryopreserved following established protocol. Sperm motility and plasma membrane integrity were recorded higher (P < 0.05) in extender containing 5.0 ng/mL of ALA compared to control. Nevertheless, sperm viability, live dead ratio and chromatin integrity were observed higher (P < 0.05) in all experimental extenders with ALA compared to control. The number of abnormal sperm reduced significantly in all experimental extenders having ALA. A total of 539 artificial inseminations were performed with the best evolved extender having ALA (5.0 ng/mL; 272 inseminations) and control (267 inseminations). In vivo fertility rates of buffalo semen were recorded higher (P < 0.05) with extender containing ALA (5.0 ng/mL) (58%) compared to control (46%). In conclusion, supplementing 5.0 ng/mL ALA in extender improved the post-thaw quality and in vivo fertility of cryopreserved Nili-Ravi buffalo bull semen. [ABSTRACT FROM AUTHOR]

Overwintering larvae of the beetle Dendroides canadensis produce potent antifreeze proteins to inhibit inoculative freezing and promote supercooling. We hypothesized that addition of Dendroides canadensis recombinant antifreeze proteins (DAFPs) in the extender will improve the quality and fertility of cryopreserved Nili-Ravi buffalo ( Bubalus bubalis ) sperm. The study was divided into two parts: (1) Evaluation of the effect of DAFPs on the quality of frozen-thawed buffalo bull sperm and (2) Examination of the fertilizing ability of frozen-thawed buffalo bull sperm. Semen was collected from three bulls using an artificial vagina (42 °C). Qualifying ejaculates from each bull were divided into four aliquots and diluted (at 37 °C, 50 × 10 6 sperm/mL) in tris-citric acid extender containing DAFP (at 0.1, 1.0, and 10 μg/mL), and the sperm were evaluated for important characteristics relative to a control without DAFP. D canadensis recombinant antifreeze proteins at any of the three concentrations did not affect sperm progressive motility or plasma membrane integrity (PMI), either before or after the semen was cooled to 4 °C in 2 hours. However, after 24 hours of cryostorage at −196 °C, followed by thawing at 37 °C for 30 seconds, sperm progressive motility and PMI were higher (P < 0.05) in extender containing DAFP at 10 μg/mL compared with control. The in vitro –fertilizing ability of cryopreserved (−196 °C) sperm supplemented with DAFP (10 μg/mL) was slightly higher (P = 0.098) compared with control, as assessed through in vitro cleavage rate of in vitro matured buffalo oocytes. Also, the in vivo fertility rate was evaluated by inseminating 100 buffaloes (50 inseminations per extender) 12 hours after standing heat. The fertility rate of cryopreserved buffalo bull sperm in terms of positive pregnancy at 90 days after insemination was clinically higher but remained statistically nonsignificant in extender containing DAFP at 10 μg/mL (52.0%) compared with control (43.8%). In conclusion, supplementation of 10 μg/mL of DAFP in the extender improved the motility and PMI of Nili-Ravi buffalo sperm after freeze-thawing, and yielded numerically higher, although statistically nonsignificant, in vitro cleavage, and in vivo fertility rate. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the use of duck egg yolk (DEY), Guinea fowl egg yolk (GFEY) and Indian indigenous hen (Desi) egg yolk (IDEY) in extender for improving the post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa, and compared it with commercial hen egg yolk (CHEY; control). For this purpose, two consecutive ejaculates of semen from each of two Nili–Ravi buffalo bulls were collected on 1 day each week for 5 weeks (replicates; n =5) with artificial vagina (42°C). Split pooled ejaculates, were diluted in tris-citric acid glycerol extender containing either DEY or GFEY or IDEY or CHEY at 37°C. Extended semen was cooled to 4°C in 2h and equilibrated for 4h at 4°C. Cooled semen was then filled in 0.5ml straws at 4°C and frozen in programmable cell freezer. Thawing of semen was performed at 37°C for 30s. Sperm motility, plasma membrane integrity and sperm morphology (acrosome integrity, head, mid-piece and tail abnormalities) of each semen sample were assessed at 0, 3 and 6h after thawing and incubation at 37°C. Visual motility (%) and percentage of intact plasma membranes assessed at 6h post-thaw of buffalo bull spermatozoa were highest (P <0.05) due to DEY as compared to GFEY, IDEY and control. The percentage of spermatozoa with normal acrosomes at 0, 3 and 6h post-thaw was highest (P <0.05) in DEY extender than GFEY, IDEY and CHEY. Sperm tail abnormalities (%) observed at 0, 3 and 6h post-thaw in samples cryopreserved with freezing extender having DEY were lower (P <0.05) as compared to extender containing GFEY, IDEY and CHEY. In conclusion, DEY compared to other avian yolks in extender improves the frozen-thawed quality of buffalo bull spermatozoa. [Copyright &y& Elsevier]