Honey as an Alternative to Antibiotics for Cryopreservation of Nili-Ravi Buffalo Bull Spermatozoa.

The antimicrobial properties of honey have stimulated interest in evaluating it as an alternative to antibiotics for cryopreserved buffalo semen. Acacia nilotica, Brassica campestris and Ziziphus jujuba honey were analyzed and Z. jujuba honey was found suitable in terms of quality and purity. Buffalo semen (24 ejaculates) was studied for in vitro dose tolerability to Z. jujuba honey (0.1%-1%), and up to 0.2% (v/v) was not toxic to buffalo spermatozoa. Afterward, semen from three bulls (24 ejaculates) was cryopreserved (four replicates) in tris-citric egg yolk extender supplemented with 0.1% or 0.2% honey, with or without streptomycin-penicillin (SP); extender with SP used as a control. After dilution and cooling, extender without antibiotics but with 0.2% honey was better (p < 0.05) than control in terms of sperm motility and plasma membrane integrity. After thawing, the extenders containing 0.1% honey with antibiotics and extender having 0.2% honey without antibiotics consistently yielded good results in terms of all parameters studied compared to control and other extenders. The extender containing 0.2% honey without antibiotics was better (p < 0.05) in terms of total aerobic bacterial count. In conclusion, 0.2% honey improves the post-thaw quality of buffalo spermatozoa and can replace the use of antibiotics in extender through its antimicrobial activity.

Retained Acetylated Histone Four in Bull Sperm Associated With Fertility.

Bull fertility, ability of the sperm to fertilize and activate the egg and support embryo development, is vital for cattle reproduction and production. Even though majority of histones are replaced by protamines, some histones are retained in sperm. It is known that chromatin remodeling during spermatogenesis results in dynamic changes in sperm chromatin structure through post-translational modifications (PTM) of sperm histones, which are important for regulation of gene expression. However, amounts of sperm Histone 4 (H4), its acetylated form (H4 acetyl), and to what extent these molecular attributes influence sperm chromatin structure and bull fertility are unknown. These gaps in the knowledge base are important because they are preventing advances in the fundamental science of bovine male gamete and improvement of bull fertility. The objective of this study was to test the hypothesis that expression dynamics as well as PTM of sperm H4 are associated with bull fertility. Flow cytometry was utilized to quantify H4 and H4 acetylated form in sperm from seven high and seven low fertility Holstein bulls. The results indicated that the average number of cells with H4 or H4 acetyl expression in high and low fertility bull sperm were 34.6 ± 20.4, 1.88 ± 1.8, 15.2 ± 20.8, and 1.4 ± 1.2, respectively. However, the sperm enriched in both H4 and H4 acetyl were different between high and low fertility groups (3.5 ± 0.6; 1.8 ± 0.8; P = 0.043). The localization and detection of H4 and H4 acetylation were measured by immunocytochemistry which revealed that H4 and H4 acetylation were equally distributed in the sperm head of high and low fertility sires. Western blotting results confirmed the presence of the H4 and its acetylated form in the sperm. Bioinformatics studies demonstrated that H4 is highly conserved among mammalians, and have significant gene ontology on spermatogenesis, early embryo implantation, and sperm capacitation. The results are significant because it demonstrates the replacement of canonical histone H4 into modified H4 acetylation in sperm and regulate its dynamics which is crucial for bull fertility and reproductive biotechnology. These findings advance fundamental science of mammalian early development and reproductive biotechnology.

Sperm cellular and nuclear dynamics associated with bull fertility.

The objective of this study was to ascertain cellular characteristics and the dynamics of the sperm chromatin proteins protamine 1 (PRM1) and protamine 2 (PRM2) in the sperm of Holstein bulls having a different fertility status. Important sperm variables were analyzed using computer-assisted sperm analysis (CASA). Sperm membrane, acrosome status, DNA integrity were also assessed using propidium iodide (PI), fluorescein isothiocyanate conjugated to Arachis hypogaea (FITC-PNA), and acridine orange (AO) followed by flow cytometry. In addition, abundances of PRM1 and PRM2 were analyzed using flow cytometry experiments. Differences in sperm decondensation capacity were assessed in bulls of varying fertility using a decondensation assay. As determined using CASA, average pathway velocity, amplitude of lateral head displacement and straightness were different (P < 0.05) for sperm from high and low fertility bulls. There, however, were no differences between the high and low fertility bulls for characteristics of sperm plasma membrane, acrosome, and DNA integrity (P > 0.05). Relative abundances of PRM1 and PRM2 in sperm from the high and low fertility bulls were inversely related (P < 0.0001). Percentages of decondensed sperm were different between high and low fertility bulls (P < 0.0001) and total numbers of decondensed sperm were greater in low fertility bulls than high fertility bulls (R2 = 0.72). Results of the present study are significant because molecular and morphological phenotypes of sperm that were detected affect fertility in livestock species.

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.

Supplementing alfa-Linolenic acid in the in vitro maturation media improves nuclear maturation rate of oocytes and early embryonic development in the Nili Ravi buffalo

The present study was conducted to investigate the effect of omega-3 poly unsaturated fatty acid (PUFA), α-linolenic acid (ALA; 18:3 n-3) on the in vitro maturation (IVM) of buffalo oocytes and subsequent embryonic development. Buffalo cumulus oocyte complexes (COCs; n = 2282) were in vitro matured in TCM-199 (0.6% fatty acid free bovine serum albumin, 0.02 Units/ml FSH, 1 µg/ml 17-β-estradiol, 10 µg/ml epidermal growth factor, 50 µg/ml gentamicin) supplemented with 0 (control), 25, 50, 100, 150 or 300 µm ALA under an atmosphere of 5% CO2 in air at 38.5ºC for 22-24 h. The matured oocytes were then fertilized in Tyrode’s Albumin Lactate Pyruvate (TALP) medium and cultured in synthetic oviductal fluid (SOF) medium. Concentrations up to 100 μm ALA improves (P ≤ 0.05) the cumulus expansion compared to control. Higher percentage of oocytes reaching MII stage was observed at 50 μm and 100 μm of ALA compared to control (P ≤ 0.05). Concentrations of 150 and 300 µm ALA were detrimental both for cumulus expansion and nuclear maturation rate of buffalo oocytes. Moreover, supplementation with 100 μm ALA improved (P ≤ 0.05) cleavage rate compared to control and treatment with 50 and 100 μm ALA yielded significantly higher morulae compared to control. The results of present study indicate that the supplementation with 100 μm ALA to the IVM medium improves nuclear maturation rate of buffalo oocytes and subsequent early embryonic development.

Supplementing alfa-Linolenic acid in the in vitro maturation media improves nuclear maturation rate of oocytes and early embryonic development in the Nili Ravi buffalo

The present study was conducted to investigate the effect of omega-3 poly unsaturated fatty acid (PUFA), α-linolenic acid (ALA; 18:3 n-3) on the in vitro maturation (IVM) of buffalo oocytes and subsequent embryonic development. Buffalo cumulus oocyte complexes (COCs; n = 2282) were in vitro matured in TCM-199 (0.6% fatty acid free bovine serum albumin, 0.02 Units/ml FSH, 1 µg/ml 17-β-estradiol, 10 µg/ml epidermal growth factor, 50 µg/ml gentamicin) supplemented with 0 (control), 25, 50, 100, 150 or 300 µm ALA under an atmosphere of 5% CO2 in air at 38.5ºC for 22-24 h. The matured oocytes were then fertilized in Tyrodes Albumin Lactate Pyruvate (TALP) medium and cultured in synthetic oviductal fluid (SOF) medium. Concentrations up to 100 μm ALA improves (P ≤ 0.05) the cumulus expansion compared to control. Higher percentage of oocytes reaching MII stage was observed at 50 μm and 100 μm of ALA compared to control (P ≤ 0.05). Concentrations of 150 and 300 µm ALA were detrimental both for cumulus expansion and nuclear maturation rate of buffalo oocytes. Moreover, supplementation with 100 μm ALA improved (P ≤ 0.05) cleavage rate compared to control and treatment with 50 and 100 μm ALA yielded significantly higher morulae compared to control. The results of present study indicate that the supplementation with 100 μm ALA to the IVM medium improves nuclear maturation rate of buffalo oocytes and subsequent early embryonic development.(AU)