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Black francolin or Black partridge (Francolinus francolinus L.) is distributed throughout Pakistan including Indus plains, Baluchistan, Punjab and Khyber Pakhtunkhwa provinces. Black francolin has been described as common or Least Concern in IUCN Red List of Threatened Species (2018). There was no documented information available on population density of Black francolin in various habitats of Kala Chitta hills of Pothwar region, Pakistan. Therefore, present study was designed to investigate the population of Black francolin in study area to devise conservation measures. Based on the reconnaissance survey, three representative sites were selected to estimate the population of black francolin in its potential habitat in Kala Chitta hills. Observations were taken by direct sighting using "Visual Encounter Method (VEM)". The observations were taken along three fixed transects, having a length of 300 m and width varying from 20 to 50 m were laid down at each site to record the birds on or near the line. Overall, population density of Black francolin was recorded 1.06 birds/ha. Highest population density (2.19 birds/ha) was recorded in mountainous forest and associated grassland. While, population density of 0.84 and 0.15 birds/ha was recorded in open cultivated land and wetlands associated vegetation, respectively. It is concluded that Black francolin population density is affected by habitat characteristics in Kala Chitta Hills. Therefore, habitat characteristics must be considered while devising conservation plan for Black Francolin. [ABSTRACT FROM AUTHOR]

The depth of intravaginal insemination to achieve optimum fertility with frozen-thawed semen is highly species specific in birds and differ even in breed and/or strains of a species. Therefore, study was designed to evaluate the influence of intravaginal insemination depths (2 and 4 cm) on fertility outcome in Indian red jungle fowl. Semen collected from eight mature cocks was pooled, diluted in extender and cooled to 4 °C. Glycerol (20%) was added to chilled semen, equilibrated for 10 min and cryopreserved. After 3 days of storage, frozen semen was thawed in water bath at 37 °C for 30 s. After glycerol removal, intravaginal Inseminations were performed at the depth of 2 and 4 cm. The no. of fertilized eggs (31.4 ± 1.6 vs. 27.7 ± 1.8), fertility rate (65.7 ± 3.6 vs. 58.8 ± 4.0), no. of hatched chicks (27.8 ± 1.9 vs. 23.5 ± 1.6), hatchability of set eggs (58.8 ± 4.3 vs. 49.7 ± 3.2) and hatchability of fertilized eggs (88.4 ± 2.8 vs. 84.3 ± 2.2) were recorded higher with intravaginal depth of 4 cm compared to 2 cm. It is concluded that intravaginal insemination at the depth of 4 cm enhances the fertility outcomes of the frozen-thawed Indian red jungle fowl semen. [ABSTRACT FROM AUTHOR]

In fragmented landscapes, the accessibility of resources not only influences the spatial distribution of the predators themselves, but may similarly affect the abundance of their prey. We studied the influence of habitat dynamics and predator density on the spatial abundance of Cape hare (Lepus capensis) in the Karakorum Range in a 5700 km2 area in Shigar valley during 2015-16 and 2017. We found that, seasonally the population density of Cape hare at deciduous forest plantation (site 3), was the highest and the lowest density of Cape hare was found in the closed to open herbaceous vegetation (site 1). Seasonally the detection probability and encounter rate were found the highest in the closed to open herbaceous vegetation (site 1), and detection probability was the lowest in the grassland and bare lands (site 2). In summer, we found a positive correlation between Cape hare and Red fox density in the deciduous forest plantation (site 3). In winter, a positive relationship between Cape hare and Red fox density were observed in the grassland and bare lands (site 2). Seasonally, the density of Cape hare were the highest recorded in the deciduous forest plantation (site 3) where the relative density of Red fox and encounter rate of active burrows were the lowest. In this economically poor study area, humans often retaliate against damages caused by local wildlife using guns. The conclusions are discussed in the perspective of predator--prey interaction and the implications for management and mitigation of human -- wildlife conflicts are also considered. [ABSTRACT FROM AUTHOR]

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]

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]

Breeding biology of Red vented bulbul (Pycnonotus cafer) was studied in the areas of Rawalpindi and Islamabad. A total of 45 nests were located in the study area out of which 35 nests were found active. Majority of successful nests (43%) were in the fork of trees followed by middle of the trees (29%) and on the top (11%) of the trees. Preferred nest height (53%) by red-vented bulbul was 1-2 meters from the ground. Vegetation material used for nest building by Red-vented bulbul was Beri (Zizyphus nummalaria) (31%) followed by Guava (Psidium guajava) (22%), Sheesham (Dalbergia sissoo) (18%), Snatha (Dodonea viscosa) (16%) and Date palm (Phoenix dactylifera) (13%). Highest percentage (51%) of nests was found with clutch size three followed by clutch size two and four. Predation rate was only 6% in eggs and 9% in nestlings. Breeding success in the present study was evaluated as good which was 82% and 86% for eggs and fledglings, respectively. Loss of nesting sites due to urban expansion and pollution negatively affect the population of Red - vented bulbul. [ABSTRACT FROM AUTHOR]

This study was carried out to determine the effect of fructose in extender on fertility of cooled buffalo semen after fixed-time insemination. Two consecutive ejaculates were collected from Nili-Ravi buffalo bull of known fertility by using artificial vagina (42°C). The pooled ejaculates were split-sampled and diluted with skimmed milk extender (37°C; 10x106 motile spermatozoa/ml) SM (control) or skimmed milk with 10mM fructose (SMF). Liquid semen was stored at 5°C and used for artificial insemination after 28 hours. Cyclic buffaloes (n=40) were randomly divided into two groups. Synchronization of estrous was accomplished by two injections of 5ml prostaglandin F2 α containing 25mg dinoprost administered 11 days apart. Each buffalo group was inseminated with either SM or SMF after 72 hours of second injection of prostaglandin F2 α. Fertility rate (%) assessed by rectal palpation 90-days after insemination was determined for two experimental extenders. The fertility rates for SM vs. SMF were 25% vs. 40%. SMF significantly (P<0.05) improved the fertility rate in buffaloes after artificial induction of heat. In conclusion, 10mM fructose in skimmed milk extender improved the fertility of chilled buffalo bull semen after artificial induction of heat. [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the feeding habits of common quail (Coturnix coturnix) in rural areas of Rawalpindi, Pakistan during September 2008 to May 2009. A total of 28 birds were captured (12 males and 16 females), gizzards were removed for feeding analysis. Out of 28 samples, two gizzards were empty and the remaining 26 samples contained 84.30% plant seeds viz., millet (Pennisetum americanum; 17.9%), sorghum (Sorghum bicolor; 17.2%), ground nut leaves (Arachis hypogaea; 10%), pohli (Carthamus oxyacantha; 9.8%), swank (Echinochloa crusgalli; 8.7%), baru (Sorghum halepense; 6.6%), and sesame (Sesamum indicum; 1%). The debris and stone contents in gizzards were 10.6 and 5.20%, respectively. The results of this study indicated that plant contents represented the major food items of the common quail in Rawalpindi, Pakistan. [ABSTRACT FROM AUTHOR]

The article discusses a research that aims to find out the prevalence of pathogenic bacterial species in black rat, Rattus rattus, house mouse, Mus musculus, and mongoose, Herpestes javanicus, at poultry farms in Pakistan's Rawalpinidi and Islamabad. About 67 specimens were captured to check the presence of bacterial species in fecal matter urine and blood of these species. The back rat's and house rat's fecal matters were contaminated with Escherichia coli, Salmonella spp., and Proteus spp. Mongoose urine was found to have Klebsiella spp. The blood samples of all species were found negative for these bacterial species.

The article discusses a study which investigated the prevalence of pathogenic bacteria in fecal matter, urine and blood of common shrew in poultry farms in Pakistan. Common shrew refers to a rodent that causes damage to the food, food products and stored products. Findings revealed that Escherichia coli and Proteus spp. was 61.5% and 15.38% in fecal matter. Meanwhile, Salmonella spp. was 69.2% in fecal matter and urine. The study concluded that common shrews act as reservoir of pathogenic bacteria which can be addressed by adopting strategic to eliminate the shrew populations.

The article discusses a study which ivestigated the prevalence of pathogenic bacteria in fecal matter, urine and blood of bandicoot captured with live traps from poultry farms of Rawalpindi and Islamabad in Pakistan. The bandicoot rats were examined for presence of bacterial species. Results show that the fecal matter were contaminated with Escherichia coli, Klebsiella pp., Proteus spp. and Salmonella spp. It concluded that the animal serves as a reservoir of bacterial infection of Escherichia coli, Klebsiella spp., Proteus spp. and Salmonella spp. at poultry farms.

Background: Egg yolk is inevitably associated with risks of microbial contamination and anti-cryoprotectant agents that necessitate the investigation of some synthetic alternatives., Objective: To investigate the potential of carboxylated poly-L-lysine (CPLL) as a replacement for egg yolk during the cryosurvivability of Nili-Ravi buffalo sperm., Materials and Methods: Semen collected from four Nili-Ravi buffalo bulls (two ejaculates / bull / day; total 40 ejaculates for five replicates) was cryopreserved in different experimental extenders viz: Control (CPLL 0%, egg yolk 20%); E1 (CPLL 5%, egg yolk 15%); E2 (CPLL 10%, egg yolk 10%); E3 (CPLL 15%, egg yolk 5%) and E4 (CPLL 20%, egg yolk 0%). Post-thaw quality was assessed in terms of sperm motility, plasma membrane integrity (PMI), viability, live:dead ratio, lipid peroxidation of sperm and total antioxidant capacity of seminal plasma., Results: Sperm motility improved (P<0.05) in extenders replacing 5%, 10% and 15% egg yolk with CPLL. Sperm PMI, viability and live:dead ratio also improved (P<0.05) in extenders replacing 10%, 15% and whole (20%) egg yolk with CPLL. In contrast, sperm DNA integrity was not different (P>0.05) when CPLL replaced egg yolk at any level. The lipid peroxidation level decreased with a concomitant increase in total antioxidant activity of seminal plasma when CPLL replaced egg yolk at 5%, 10%, 15% and 20%., Conclusion: Replacement of 15% egg yolk in the extender with CPLL improves all sperm quality parameters: motility, PMI, viability, live:dead ratio, lipid peroxidation of sperm and total antioxidant activity of seminal plasma.

During cryopreservation sperm encounter oxidative stress due to higher production of ROS molecules and insufficient natural antioxidant defence system. Therefore, present study was designed to identify the effects of various glutathione (GSH) concentrations on Indian red jungle fowl (Gallus gallus murghi) sperm quality and fertility pre-freezing and post-thaw incubation hours. Semen was collected from eight cocks and qualified semen ejaculates having motility >65% were pooled after initial evaluation. Semen was divided in four aliquots, diluted with red fowl extender (1:5) at 37 °C having GSH 0 mM (control), 0.1 mM, 0.5 mM and 1.0 mM, cryopreserved and stored at (-196 °C) in liquid nitrogen. Semen quality was assessed at post dilution, cooling, equilibration, and freeze-thawing at 0, 2 and 4 h of incubation at 37 °C. Sperm motility, plasma membrane integrity, viability, acrosome integrity and mitochondrial function were recorded highest (P < 0.05) with 0.5 mM GSH in extender at post-dilution, cooling, equilibration, freeze-thawing and 0, 2 and 4 h of incubation. Lipid peroxidation in sperm and seminal plasma were recorded lowest (P < 0.05) with 0.5 mM GSH during cryopreservation stages and post-thawing incubation. Moreover, antioxidant activities (total antioxidant potential and free radical scavenging capacity) were recorded highest (P < 0.05) in extender having 0.5 mM GSH. Fertility rates were recorded higher (P < 0.05) with 0.5 mM GSH compared to control. It is concluded that 0.5 mM GSH in extender improves sperm structural (sperm viability, plasma membrane integrity and acrosome integrity), functional integrity (motility, mitochondrial function) and fertility parameters of Indian red jungle fowl through enriching antioxidant potential and ameliorating the oxidative stress., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The present study investigates the efficacy of dimehtlyformamide (DMF) as a permeable cryoprotectant and its effect on quality and fertility of Indian red jungle fowl sperm. Semen was collected from eight mature roosters, pooled, divided into five aliquots and diluted with red fowl extender having DMF (0%, 4%, 6%, 8% and 10%). Diluted semen samples were cooled from 37 °C to 4 °C, 20% glycerol added to control (0% DMF), equilibrated for 10 min and filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Sperm motility, plasma membrane functionality, viability and acrosome integrity were assessed at post dilution, cooling, equilibration and freeze-thawing stage of cryopreservation. Cryopreservation stages had negative effects (P < 0.05) on semen quality parameters. Percentages of sperm motility, plasma membrane functionality, viability and acrosome integrity were recorded highest in extender having 8% DMF at post-dilution, cooling, equilibration and freeze-thawing stage. Fertility results after artificial insemination were recorded higher (P < 0.05) with 8% DMF compared to 20% glycerol. Dimehtlyformamide (8%) in red fowl extender improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The quality of cryopreserved buffalo semen is low due to high susceptibility of sperm membranes to cold shock., Objective: The present study was designed to investigate the effect of recombinant type-III antifreeze protein from the eel pout Macrozoarces americanus (rAFPIII) on freezability of buffalo semen., Materials and Methods: Semen was collected from three buffalo bulls for three weeks (replicates). Qualified ejaculates (N=18) were split into four aliquots and diluted in Tris-citric acid extender containing 0.0, 0.1, 1 and 10 µg mL -1 of rAFPIII. Semen was cooled to 4 C, evaluated for sperm motility and PMI, cryopreserved and assessed for post-thaw quality., Results: Supplementation of the extender with rAFPIII didn't affect motility and PMI of chilled semen. Post-thaw sperm motility and PMI were higher in extender supplemented with rAFPIII (10µg mL -1 ) compared to control. Sperm viability and acrosome integrity remained the same., Conclusion: Addition of rAFPIII in extender improved motility and PMI of cryopreserved buffalo semen.

Glycerol is a least toxic and most effective cryoprotectant for cryopreservation of poultry semen, but due to its contraceptive properties removal of glycerol is usually needed prior to artificial insemination. Dimethylsulfoxide (DMSO), a small amphiphilic molecule used as penetrating cryoprotectant for biological cells, has been recognized as an adequate alternative for cryopreservation of sperm from several species. This study was designed to evaluate the efficacy of different concentrations of DMSO as cryoprotectant for Indian red jungle fowl (Gallus gallus murghi) sperm. Semen was collected from Indian red jungle fowl cocks, pooled and divided into five aliquots. Different concentrations of DMSO (0%, 4%, 6%, 8% and 10%) were compared. Diluted semen was cooled from 37 °C to 4 °C (-0.275 °C min -1) , 20% glycerol added to control and equilibrated for 10 min. After equilibration, semen was filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Semen samples were thawed at 37 °C for 30 s. Cryo-survival of Indian red jungle fowl sperm was affected by cryopreservation stages and different concentrations of cryoprotectant used. Highest sperm motility (85.0 ± 2.9; 80.0 ± 3.5; 71.3 ± 4.3; 60.0 ± 1.3), plasma membrane integrity (79.5 ± 3.8; 75.3 ± 2.4; 72.8 ± 3.3; 60.3 ± 2.8), viability (80.8 ± 4.6; 75.5 ± 2.9; 71.0 ± 7.6; 58.8 ± 1.3) and acrosomal integrity (76.3 ± 2.4; 72.0 ± 6.0; 62.5 ± 4.3; 55.0 ± 3.2) were recorded in a diluent having 8% DMSO at post-dilution, cooling, equilibration and freeze-thawing. Highest fertility results were obtained after artificial insemination with 8% DMSO compared to 20% glycerol (73.0 ± 4.4 vs 53.1 ± 4.3, P < 0.05). It is concluded that 8% DMSO as a permeable cryoprotectant improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Egg yolk is a good external cryoprotectant of mammalian sperm and some wild bird's sperm, but, at least in domestic breeds of chicken (Gallus gallus domesticus), it may inhibit eventual fertilization of ova when high concentrations are used. We hypothesized that egg yolk can protect the sperm from cryo-induced damages providing adequate fertilization in one phylogenetic wild ancestor of current chicken breeds: the Indian red jungle fowl (Gallus gallus murghi). To test the hypothesis, the present study was designed to evaluate different concentrations of egg yolk in extender in comparison with glycerol. Semen collected from Indian red jungle fowl cocks (n = 8) was cryopreserved using different egg yolk concentrations (10%, 15%, 20% and 25%) or 20% glycerol (control group) following routine protocol of cryopreservation. During cryopreservation, sperm motility (67.5 ± 2.5%), plasma membrane integrity (66.3 ± 2.4%), viability (58.8 ± 1.3%) and acrosomal integrity (60.0.8 ± 2.0%) were recorded highest in an extender with 15% egg yolk compared to other experimental extenders and control at post-dilution, cooling, equilibration and thawing. The no. of fertilized eggs (26.6 ± 0.7, 21.6 ± 1.2), percent fertility (55.9 ± 4.4, 46.5 ± 2.2), no. of hatched chicks (23.6 ± 1.0, 17.2 ± 1.0), percent hatch (49.5 ± 3.2, 37.1 ± 2.5%) and hatchability of the fertile eggs (89.4 ± 2.2, 79.7 ± 3.7) were recorded higher (P < 0.05) with semen cryopreserved with 15% egg yolk compared to control (20% glycerol). It is concluded that 15% egg yolk can be used in cryopreservation protocol of Indian red jungle fowl sperm., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 × 10 6 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, cryopreserved in extenders containing 15% PEY or 20% chicken egg yolk was assessed for fertility after artificial inseminations. 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., (Copyright © 2018. Published by Elsevier Inc.)

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 × 10 6 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., (© 2017 Blackwell Verlag GmbH.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

It was hypothesized that dimethyleacetamide (DMA) can be used as an alternate to glycerol for cryopreservation of Indian red jungle fowl semen. Four concentrations of DMA (4%, 6%, 8% and 10%) in extender were compared with previously optimized cryopreservation protocol based on 20% glycerol (control) for Indian red jungle fowl. Sperm motility, plasma membrane integrity, viability, and acrosome integrity were assessed at the stage of post-dilution, cooling, equilibration, and freeze-thawing. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) at post-dilution, cooling, equilibration, and freeze-thawing in extender having 6% DMA compared to control and other experimental extenders. The highest (P < 0.05) recovery rates of all aforementioned parameters were also recorded in extender having 6% DMA; thus, 6% DMA was further compared with control (20% glycerol) for fertility after artificial insemination. Eggs were collected for five days after artificial insemination with semen cryopreserved in extender containing 6% DMA and control. The higher no. of fertilized eggs, fertility, no. of hatched eggs, hatch (%) and hatchability were recorded with semen cryopreserved in extender having 6% DMA compared to control. It is concluded that 6% DMA maintained higher post-thaw quality and fertility of Indian red jungle fowl semen and is a better replacement of glycerol., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 a retrospective manner. Semen ejaculates (n = 1148) were regularly collected from eight adult cocks 6 to 54 months of age. Quantitative and qualitative semen parameters viz; volume (μL), concentration (1 × 10 9  mL -1 ), total sperm number per ejaculate (1 × 10 9  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 and semen quality factor) was observed. The highest values were observed at four years of age (P < 0.05) with the exception of sperm motility that was not affected by the age. Spring was the best season for sperm parameters viz; volume, motility, plasma membrane integrity, viability and acrosomal integrity (P < 0.05), however a remarkable sperm production was noticed all over the year. It is concluded that Indian red jungle fowl exhibits an evolution of sperm production that greatly differs in many points from other fowl sub-species. It is suggested that semen ejaculates of highest quality achieved for semen banking at the age of four year in the spring season., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Semen from the Nili-Ravi buffalo bull, Bubalus bubalis, shows poor survival after freeze storage compared to bovine (Bos taurus and Bos indicus) semen. Freeze-susceptibility distinctions in these two genera have been attributed to differences in sperm membranes., Materials and Methods: We measured the impact of protein preparations derived from a frost-resistant perennial grass, Lolium perenne, with ice recrystallization inhibition activity on the low temperature storage of B. bubalis semen., Results: When the L. perenne preparations (0.1, 1, 10 µg/mL) were added to buffalo semen [2 ejaculates per bull (N=3) per replicate (r=3)] in Tris-citrate extender (50×10 6 sperm mL -1 ), there was no impact on semen quality, as measured by sperm motility and plasma membrane integrity, after storage at 4 degree C (P>0.05). However, when semen supplemented with the grass proteins (0.1 and 1 µg mL -1 ) was evaluated after freezing and storage in liquid nitrogen for 24 h, post-thaw sperm progressive motility and plasma membrane integrity was higher (P<0.05) than in control samples. Post-thaw sperm viability and sperm acrosome integrity was similar (P > 0.05) to controls., Conclusion: The improvement in cryopreserved buffalo sperm progressive motility and plasma membrane integrity suggests that the use of these easily-made preparations may improve fertility after cryopreservation and offers the prospect of improved conception rates after artificial insemination with cryopreservation.

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 × 10 6 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 < 0.05) in extender containing 5% QEY compared with control. However, TEY at 10% in extender improved (P < 0.05) the post-thaw sperm motility (57.5 ± 5.2), plasma membrane integrity (53.5 ± 4.5), livability (75.3 ± 6.0), and viability (73.5 ± 6.5) compared with higher concentrations of TEY and controls (20% CEY). The chromatin damage (2.0 ± 0.9) and intracellular enzymes, glutamic oxaloacetic transaminase (24.8 ± 3.5) and lactic dehydrogenase (77.7 ± 4.5), release were lower (P < 0.05) in extender containing 10% TEY compared with the controls. Invivo fertility was compared after artificial insemination with semen from two buffalo bulls that was cryopreserved in extenders containing 5% QEY, 10% TEY, or 20% CEY. A total of 600 inseminations (200 inseminations per extender) were recorded; the overall fertility rate was significantly higher (P < 0.05) with semen cryopreserved in extender containing 5% QEY (57.5 vs. 42%) and 10% TEY (57.5 vs. 42%). compared with 20% chiken egg yolk. In conclusion, QEY at 5% and TEY at 10% offers advantages over 20% CEY in terms of in vitro post-thaw semen quality and in vivo fertility of buffalo., (Copyright © 2016. Published by Elsevier Inc.)

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 10min, filled in 0.5mL French straws, kept over LN 2 vapours for 10min 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 4h 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016. Published by Elsevier Inc.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Experiments were conducted to evaluate the effect of Antarctic fish antifreeze glycoproteins, (AFGP) size 1-5 (34-10.5 kDa) and 7-8 (3.2 and 2.4 kDa) in extender on buffalo bull sperm at cooling (4 °C) and at post thawing. Semen was collected from three Nili-Ravi buffalo bulls with artificial vagina for 3 weeks. Qualifying ejaculates from each buffalo bull were diluted (at 37 °C having 50×10(6) sperm/mL) in tris-citric acid extender containing AFGP at 0 (control), 0.1, 1 and 10 μg/mL. An aliquot of diluted semen was evaluated for sperm progressive motility and plasma membrane integrity, while the remaining fraction was cooled to 4 °C in 2 h. Further, an aliquot of cooled semen was evaluated for the previously described variables and the remaining fraction was cryopreserved (-196 °C). After 24 h of storage, straws were thawed at 37 °C for 30 s to assess post-thaw sperm quality. Inclusion of AFGP in the extender did not affect (P>0.05) sperm progressive motility and plasma membrane integrity of buffalo bull sperm at cooling stage (4 °C). However, at post thawing, improvement (P<0.05) in sperm progressive motility and plasma membrane integrity was recorded in extender containing AFGP 1-5 and AFGP 7-8 at 1 μg/mL compared to the control. Percentage of live sperm with an intact acrosome remained similar (P>0.05) in extenders containing different amounts of AFGP and control. In conclusion, supplementation of 1 μg/ml of AFGP in extender improved the motility and plasma membrane integrity of Nili-Ravi buffalo sperm after thawing., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lower fertility in buffaloes with frozen-thawed semen is attributed to sperm damage that is believed to be due to formation of ice crystals during freeze/thaw process. It was hypothesized that antifreeze proteins in the extender may improve the post thaw quality of buffalo bull sperm. For this purpose, two separate experiments were conducted to evaluate antifreeze proteins III (AFP III) at 0 (control), 0.1, 1 and 10 μg/mL (Experiment I) and 0 (control), 0.01, 0.1 and 1 μg/mL (Experiment II) for its effect on post thaw quality of buffalo bull semen. Semen was collected from three Nili-Ravi buffalo (Bubalus bubalis) bulls with artificial vagina (42 °C) for three weeks (replicate) per experiment. For each experiment, qualifying ejaculates (6 ejaculates/bull) were divided into four aliquots and diluted (at 37 °C having 50 × 10(6) sperm/mL) in tris-citric acid extender containing above mentioned concentrations of AFP III. Diluted semen was cooled to 4 °C in 2 h, equilibrated for 4 h, filled in 0.5 mL straws, kept over liquid nitrogen vapors for 10 min and plunged in the liquid nitrogen. After 24 h of storage, semen straws were thawed at 37 °C for 30 s to assess sperm progressive motility (SM), plasma membrane integrity (PMI), viability (live sperm with intact acrosome) and normal epical ridge (NAR). In experiment I, improvement (P<0.05) in percentage SM and sperm PMI was recorded in extender containing 0.1 μg/mL AFP III compared to control, the higher concentrations (1 μg/mL and 10 μg/mL) being inefficient. While evaluating the lower concentration (experiment II), 0.01 μg/mL of AFP III in the extender it was found to be ineffective to improve semen quality parameters, while 0.1 μg/mL AFP III in extender was found better in terms of progressive motility and plasma membrane integrity of buffalo bull semen compared to control. Sperm viability and NAR remained similar (P>0.05) in extenders containing different concentrations of AFP III and control in both of experiments. In conclusion addition of AFP III in the extender at 0.1 μg/mL improved the progressive motility and plasma membrane integrity of cryopreserved buffalo bull semen., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post-thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post-thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili-Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris-citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 10(6) spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5-ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose-dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome-intact live sperm (%) and sperm chromatin integrity (%) were better (p < 0.05) in extender having 5.0 ng/ml of arachidic acid compared with control. At 10.0 ng/ml, these values did not vary (p > 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome-intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post-thaw quality parameters of cryopreserved Nili-Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post-thaw semen quality parameters was observed at 20.0 ng/ml., (© 2013 Blackwell Verlag GmbH.)

Ciprofloxacin (CP) was evaluated for bacterial control, post-thaw quality, and fertility of buffalo semen. Pseudomonas aeruginosa, Escherichia coli, Proteus sp., Corynebacterium sp., Micrococcus sp., and Staphylococcus sp. were isolated from buffalo semen. Pseudomonas aeruginosa, Corynebacterium sp., and Micrococcus sp. were resistant to streptomycin, whereas P. aeruginosa and Proteus sp. were resistant to penicillin. All bacteria were susceptible to CP. In vitro dose toxicity was assessed in sodium citrate buffer containing 0, 200 to 2000 μg/mL of CP. CP up to 1000 μg/mL was found nontoxic to motility and viability of buffalo sperm. For post-thaw quality, buffalo semen was frozen in Tris-citric acid extender containing streptomycin-penicillin (SP; 1000 μg/mL-1000 IU/mL) or CP 600 μg/mL and was assessed for total aerobic bacterial count (post-thaw), motility, plasma membrane integrity, viability at 0, 2, and 4 hours post-thaw. At 4 hours post-thaw, plasma membrane integrity (%) was higher (P < 0.05) in extender containing CP than SP. Total aerobic bacterial count was 0.00 in extender containing CP compared with 0.07 × 10(4) cfu/mL with SP. To assess the in vivo fertility rate, semen (two bulls) frozen in Tris-citric acid extender containing SP or CP was used to inseminate, and 400 inseminations (200/group) were recorded. Higher (P ≤ 0.05) fertility rate was recorded with CP (55%) compared with SP (41%). In conclusion, use of CP in extender was efficient to control the bacterial contamination without compromising the post-thaw quality and fertility of cryopreserved water buffalo bull semen., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen., (© 2011 Blackwell Verlag GmbH.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study was designed to compare the quality of liquid-stored buffalo bull spermatozoa in soya lecithin based extender Bioxcell(®) (BIOX), milk (MILK), tris-citric egg yolk (TEY) and egg yolk-citrate (EYC) extender at 5°C. Semen was collected from five Nili-Ravi buffalo (Bubalus bubalis) bulls of 6-7 years of age with artificial vagina over a period of 3 weeks (two consecutive ejaculates once in a week). Semen ejaculates having more than 60% motility were pooled, split into four aliquots, diluted (37°C; 10 × 10(6) motile spermatozoa/ml), cooled from 37 to 5°C in 2 h (0.275°C/min) and stored for 5 days. Sperm motility, viability, plasma membrane integrity (PMI) and normal acrosomal ridge were studied at first, third and fifth day of storage. Higher values of progressive sperm motility (%), sperm viability (%), sperm PMI (%) and normal apical ridge (%) were observed in BIOX, MILK and TEY extenders at first, third and fifth day of storage than EYC extender. Progressive sperm motility, sperm viability and sperm PMI in BIOX(®) extender were not different from MILK and TEY extenders at 1st and third day storage period. However, at fifth day of storage, the values for these parameters remained significantly higher (p<0.05) in BIOX(®) compared with MILK, TEY and EYC extenders. At fifth day of storage, the semen quality parameters for Bioxcell(®) were comparable to those with MILK and TEY extenders at third day of storage. In conclusion, motility, viability and PMI of buffalo bull spermatozoa remained similar in Bioxcell(®) , milk and TEY extender at first and third days of storage at 5°C. Yet, the values for the aforementioned parameters in Bioxcell(®) were higher compared with milk, TEY and EYC extender at fifth day of storage at 5°C., (© 2010 Blackwell Verlag GmbH.)

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 motility >60% 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 (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., ((c) 2010 Elsevier Inc. All rights reserved.)