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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.

Egg yolk is used as a cryoprotectant in semen preservation. However, its composition varies according to the species which may influence its effectiveness during the freeze-thaw process. Therefore, study was conducted to identify the optimum level of pigeon egg yolk (PEY) in Tris citric acid (TCA) extender for freezability and in vivo fertility of buffalo semen. Semen was collected at weekly intervals for a period of three weeks (replicates) from 6 Nili Ravi buffalo bulls (2 ejaculates/bull/ replicate) and diluted with TCA extender (50 × 106 motile spermatozoa ml-1) containing 5%, 10%, 15% and 20% PEY or 20% CEY (control) and cryopreserved. Post-thaw sperm quality and extracellular enzymes leakage was assessed after thawing. Sperm motility, plasma membrane integrity, livability and viability was significantly higher in extenders containing 10% and 15 % PEY compared to 5% PEY, 20% PEY or 20 % CEY (controls). A dose-dependent decrease was recorded in the chromatin damage for the PEY, being lowest for the 15% and 20% PEY which was significantly less compared to controls (20%CEY). The extracellular GOT and LDH leakage was significantly lower (P < 0.05) in extender containing 10% and 15 % PEY compared to the controls. Semen collected from 2 bulls and cryopreserved in extenders containing 15% PEY or 20% chicken egg yolk was assessed after AI. A total of 400 buffaloes were inseminated (100 inseminations/extender/bull). The overall fertility rate was significantly higher (P < 0.05) with semen cryopreserved in extender containing 15% PEY (56 %) compared to 20% CEY (42 %; controls). In conclusion, pigeon egg yolk at 15 % offers advantages over 20% chicken egg yolk in terms of in vitro post-thaw semen quality and in vivo fertility of buffalo.

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.

The reproductive potential of the adult males is expected to vary with age/season and largely differ not only in closely related avian species but even in subspecies, breeds and/or strains of the same species. Thus, it is pre-requisite to have knowledge of seminal parameters to achieve maximum production potential of at-risk species for ex situ in vitro conservation programs. A 4-year study was designed to evaluate the effect of age and season (spring, summer, autumn and winter) on semen characteristics of Indian red jungle fowl (Gallus gallus murghi) in retrospective manner. Semen ejaculates (n=1148) were regularly collected from eight adult Indian red jungle fowls from 6 to 54 months of age. Quantitative and qualitative semen parameters viz; volume (μL), concentration (1x109 mL−1), total sperm number per ejaculate (1x109 mL−1), motility (%), viability (%), plasma membrane integrity (%), acrosome integrity (%) and semen quality factor were recorded. A chronological increasing trend with age of most sperm quantitative and qualitative traits (semen volume, sperm concentration, total sperm number per ejaculate, plasma membrane integrity, viability, acrosomal integrity, semen quality factor) was observed. The highest values were observed at four years of age (P

Egg yolk is used as a cryoprotectant for semen in different mammalian species including buffalo. Egg yolk from different sources may affect freezability of buffalo bull semen. Quail egg yolk (QEY) and turkey egg yolk (TEY) in Tris-citric acid extender was evaluated for post-thaw quality and in vivo fertility rate of cryopreserved buffalo bull semen. Ejaculates were collected on weekly basis from six Nili-Ravi buffalo bulls (12 ejaculates/bull) for a period of 6 weeks and diluted at 37 °C with tris-citric egg yolk extender (50 × 106 motile spermatozoa mL−1) containing different levels of QEY or TEY (5%, 10%, 15%, and 20%) or 20% chicken egg yolk (CEY; controls) and cryopreserved. Percent post-thaw sperm motility (48.3 ± 3.8), plasma membrane integrity (67.9 ± 5.3), live/dead ratio (68.2 ± 5.0), and viability (50.5 ± 3.7) were recorded higher (P

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.

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 × 10(6) 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.

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 degrees C) for three weeks (replicates). Qualifying ejaculates having motility60% from each buffalo bull were divided in two aliquots and diluted (at 37 degrees C having 50 x 10(6) spermatozoa/ml) in tris-citric egg yolk or Bioxcell extender. Diluted semen was cooled to 4 degrees 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 degrees C). After 24 hours of storage, semen straws were thawed at 37 degrees 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 (P0.05) 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 (P0.05) 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.