Efficiency of beetle (Dendroides canadensis) recombinant antifreeze protein for buffalo semen freezability and fertility.

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.

Qadeer S ，Khan MA ，Shahzad Q ，Azam A ，Ansari MS ，Rakha BA ，Ejaz R ，Husna AU ，Duman JG ，Akhter S ... -  《-》

Evaluation of antifreeze protein III for cryopreservation of Nili-Ravi (Bubalus bubalis) buffalo bull sperm.

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.

Qadeer S ，Khan MA ，Ansari MS ，Rakha BA ，Ejaz R ，Husna AU ，Ashiq M ，Iqbal R ，Ullah N ，Akhter S ... -  《-》

Efficiency of antifreeze glycoproteins for cryopreservation of Nili-Ravi (Bubalus bubalis) buffalo bull sperm.

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.

Qadeer S ，Khan MA ，Ansari MS ，Rakha BA ，Ejaz R ，Iqbal R ，Younis M ，Ullah N ，DeVries AL ，Akhter S ... -  《-》

Cryopreservation of Nili-Ravi buffalo (Bubalus bubalis) semen in AndroMed(®) extender; in vitro and in vivo evaluation.

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.

Ansari MS ，Rakha BA ，Akhter S 《-》

Royal jelly supplementation in semen extender enhances post-thaw quality and fertility of Nili-Ravi buffalo bull sperm.

Two experiments were conducted to evaluate the effect of royal jelly (RJ) on post-thaw sperm quality, in vitro and in vivo fertility rate of cryopreserved buffalo bull sperm. The semen was collected from three mature regular donor buffalo bulls, ejaculates were pooled and semen evaluated initially. In Experiment 1, the ejaculates were extended in tris-citric acid diluter supplemented with different RJ concentrations (0, 0.05, 0.1, 0.2, 0.3 or 0.4%). The diluted semen was cooled to 4°C, packaged into 0.5 mL straws and frozen using standard procedure. The straws were thawed and assessed for sperm progressive motility, viability, plasma membrane, acrosome, and chromatin integrity. The results indicated that sperm progressive motility was significantly greater (P<0.05) in 0.05, 0.1, 0.2 and 0.3% RJ than 0.4% RJ supplemented and control groups. The sperm viability, plasma membrane and acrosome integrity were significantly improved (P<0.05) in 0.1% RJ supplemented group the compared to other treatment groups. In Experiment 2, cryopreserved sperm with 0.1% RJ supplementation and control (without RJ supplementation) were used to observe the in vitro fertilizing potential and in vivo fertility. In vitro fertilization method was applied to assess the cleavage rate; whereas, AI was performed in buffalo during in vivo fertility trial. The buffaloes were inseminated 12h after standing estrus and pregnancy diagnosis was performed through ultrasonography. The results revealed that the cleavage rate was higher (P<0.05) in 0.1% RJ as compared to control group. However, the pregnancy rate was similar (P>0.05) between 0.1% RJ supplemented and control groups. It is concluded that supplementation of RJ in freezing extender can improve the cryosurvival rate and in vitro fertilizing capacity of buffalo bull sperm.

Shahzad Q ，Mehmood MU ，Khan H ，ul Husna A ，Qadeer S ，Azam A ，Naseer Z ，Ahmad E ，Safdar M ，Ahmad M ... -  《-》