J.P. Ravindra

Effect of Taurine and Melatonin in the Culture Medium on Buffalo In Vitro Embryo Development

This study was carried out to investigate the effect of supplementing culture medium with different concentrations of taurine and melatonin, on buffalo oocyte in vitro meiotic maturation and embryo development. In experiment 1, oocytes were matured in vitro and the cleaved embryos were cultured in the same following seven culture medium; (i) control (TCM 199 + 10% SS); (ii) control + 0.5 mM taurine; (iii) control + 1 mM taurine; (iv) control + 3 mM taurine; (v) control + 5 microM melatonin; (vi) control + 10 microM melatonin and (vii) control + 50 microM melatonin. In experiment 2, based on the results of experiment 1, to examine the synergistic effect of antioxidants, the oocytes were matured in culture medium (TCM199 + 10% SS), supplemented with both taurine at 1 mM and melatonin at 10 microM concentration and the cleaved embryos were cultured in the same medium. Supplementation of taurine at 1 mM concentration in the culture medium resulted in a higher (p < 0.05) transferable embryo (TE) yield when compared with control (20.6% vs 14.1%). Supplementation of melatonin at 10 and 50 microM concentration in the culture medium resulted in a higher (p < 0.05) meiotic maturation rate (90.3% and 88.8% respectively) and TE yield (28.4% and 27.2% respectively), than the other treatments. In experiment 2, the TE yield did not improve by supplementing the culture medium with both taurine and melatonin, when compared with melatonin alone. In conclusion, the results of this study demonstrated that, enriching the culture medium with taurine and melatonin, improves in vitro embryo production efficiency in buffaloes. In particular, a high TE yield was obtained by enriching the culture medium with 10 microM melatonin.

Influence of IGF-I on buffalo (Bubalus bubalis) spermatozoa motility, membrane integrity, lipid peroxidation and fructose uptake in vitro

The objective of the present experiment was to examine the influence of mean physiological concentration of insulin-like growth factor-I (IGF-I) on frozen-thawed Surti buffalo (Bubalus bubalis) spermatozoa functional parameters, i.e., motility, plasmalemma integrity, acrosomal integrity, functional membrane integrity, lipid peroxidation and fructose uptake in vitro. Frozen-thawed semen samples (n=6) were washed in tris buffer and divided into two equal parts (control and IGF-I groups). Only in the IGF-I group, IGF-I (rhIGF-I analogue) was added to a final concentration of 100 ng/ml. The samples were incubated at 37 degrees C for 2h and the assessments were made at 0, 30, 60, 90 and 120 min of incubation. The mean concentration of the buffalo seminal plasma (n=17) IGF-I was 116.83+/-28.34 ng/ml (range 41.4-198.95). IGF-I had significant effect on the total motility (P<0.01), progressive forward motility (P<0.01), functional membrane integrity (P<0.05) and lipid peroxidation levels (P<0.05) during the 120-min study period as assessed by area under curve. Treatment with IGF-I increased (P<0.01) the total spermatozoa motility at 30, 60 and 90 min as compared to the control. The progressive forward motility was significantly (P<0.01) higher at 60 and 90 min of incubation. The addition of IGF-I resulted in significant (P<0.01) increase in straight-line velocity (VSL, microm/s) as compared to the control at 60 and 90 min of incubation. The linearity (%) was significantly (P<0.01) higher in IGF-I treated semen as compared to control at 60 min of incubation. Plasmalemma integrity in IGF-I group was significantly (P<0.05) higher than control at 30 and 60 min of incubation. The functional membrane integrity differed significantly (P<0.01) between groups (control and IGF-I) at 60 and 90 min of incubation. The percentage of acrosomal intact spermatozoa decreased continuously over a period of time in both the groups. As compared to 0 min of incubation, the significant (P<0.05) loss of acrosome was observed at 60 and 90 min of incubation in control (63.87+/-3.17 vs. 58.52+/-2.54) and IGF-I (61.60+/-2.26 vs. 56.11+/-2.12) groups, respectively. Lipid peroxidation levels were significantly lower in IGF-I group at 90 min (P<0.05) and 120 min (P<0.01) of incubation than the control group. Fructose utilization was significantly higher in IGF-I group as compared to control at 30 min (P<0.05) and 60 min (P<0.01) of incubation. The present study suggests that addition of IGF-I improve spermatozoa functional parameters by reducing lipid peroxidation levels.

Effect of breeding season on in vivo oocyte recovery and embryo production in non-descriptive Indian river buffaloes (Bubalus bubalis)

The present study was carried out to examine the effect of season on in vivo oocyte recovery and embryo production in non-descriptive, Indian river buffaloes (Bubalus bubalis). Ovum pick up (OPU) was conducted twice a week for 8 weeks during peak (October-March) and low (April-September) breeding season in live buffaloes (n=6). OPU was performed using ultrasound equipment with a 5MHz transvaginal transducer, a single lumen 18-gauge, 55-cm long needle and a constant vacuum pressure of 110mmHg. The number and size of follicles was determined before puncture. The recovered oocytes were graded and only grade A and grade B oocytes were used for in vitro production (IVP) of embryos. The mean number of follicles observed per animal per session did not differed (P<0.05) between animals or between puncture sessions in both low and peak breeding seasons. Higher (P<0.05) number of follicles were observed (4.8+/-0.2 versus 3.1+/-0.3) and punctured (4.0+/-0.2 versus 2.4+/-0.2) during peak breeding season when compared to low breeding season. Oocytes recovered (1.6+/-0.1 versus 1.0+/-0.3) per animal per session were higher (P<0.05) in peak breeding season than low breeding season. During the peak breeding season, the blastocyst yield per animal per session (0.3+/-0.4 versus 0.18+/-0.4) was higher (P<0.05) than the low breeding season. However, season did not significantly affect the percentage of oocytes suitable for IVP (grade A+B) and blastocyst production rate. In conclusion, the efficiency of OPU combined with IVP was higher during the peak breeding season than the low breeding season in buffaloes.

Prognostic Value of Various Spermatological Attributes as Predictors of Zona Binding and Zona Penetration of Buffalo (Bubalus bubalis) Semen

Twenty-four ejaculates from six (four ejaculates each) Surti buffalo bulls aged 4-8 years were used to assess various attributes of spermatozoa influencing the zona-binding and zona-penetration tests. Ejaculates from each bulls were subjected to in vitro sperm--zona binding and sperm--zona penetration tests (four replicates per bull) using immature buffalo oocytes. The average number of spermatozoa bound per oocyte was 27.79 +/- 5.90. The average number of spermatozoa penetrated per oocyte was 3.35 +/- 0.64. The average number of zona-bound and -penetrated spermatozoa differed significantly between animals. Significant difference (p < 0.05) was observed between the plasmalemma integrity as assessed by eosin--nigrosin stain and hypo-osmotic swelling (HOS) test. Furthermore, the percentage of cells positive for the HOS test, i.e. functional membrane integrity (51.25 +/- 2.32) was significantly (p < 0.05) higher than hypo-osmotic swelling-Giemsa (HOS-G) test, i.e. the subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (42.87 +/- 4.56). The HOS test had significant correlations with plasmalemma integrity as measured by the vital stain, eosin--nigrosin (r = 0.85, p < 0.05). The HOS-G test also had significant correlation with plasmalemma integrity measured by vital stains such as eosin--nigrosin (r = 0.90, p < 0.05) and fluorogenic stains [carboxyfluorescein diacetate (CFDA) and propidium iodide (PI); r = 0.92, p < 0.01] and HOS test (r = 0.93), acrosomal integrity (r = 0.86, p < 0.05) and mitochondrial membrane potential (r = 0.99, p < 0.01). The plasmalemma integrity (fluorogenic stain), functional membrane integrity (HOS test), subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (HOS-G test) and mitochondrial membrane potential had significant (p < 0.05) correlation with sperm zona binding and penetration. The present study indicates that these parameters could represent important determinants of sperm quality influencing zona binding and penetration.

Occurrence and functional significance of the transcriptome in bovine ( Bos taurus ) spermatozoa

Mammalian spermatozoa deliver various classes of RNAs to the oocyte during fertilization, and many of them may regulate fertility. The objective of the present study was to determine the composition and abundance of spermatozoal transcripts in fresh bull semen. The entire transcriptome of the spermatozoa from bulls (n = 3) was sequenced using two different platforms (Ion Proton and Illumina) to identify the maximum number of genes present in the spermatozoa. The bovine spermatozoa contained transcripts for 13,833 genes (transcripts per million, TPM > 10). Both intact and fragmented transcripts were found. These spermatozoal transcripts were associated with various stages of spermatogenesis, spermatozoal function, fertilization, and embryo development. The presence of intact transcripts of pregnancy-associated glycoproteins (PAGs) in the spermatozoa suggest a possible influence of sperm transcripts beyond early embryonic development. The specific regions (exon, intron, and exon-intron) of the particular spermatozoal transcripts might help regulate fertilization. This study demonstrates that the use of two different RNA-seq platforms provides a comprehensive profile of bovine spermatozoal RNA. Spermatozoal RNA profiling may be useful as a non-invasive method to delineate possible causes of male infertility and to predict fertility in a manner that is more effective than the conventional methods.

Introduction to Concepts of Climate Change Impact on Livestock and Its Adaptation and Mitigation

This chapter provides an overview of the impact of climate change on livestock production and its adaptation and mitigation. Animal agriculture is the major contributor to increasing methane (CH4) and nitrous oxide (N2O) concentrations in Earth’s atmosphere. Generally there are two-way impacts of livestock on climate change. The first part is the livestock contribution to climate change, while the second part is concerned with livestock getting affected by climate change. Hence, improving livestock production under changing climate scenario must target both reducing greenhouse gas (GHG) emission from livestock and reducing the effect of climate change on livestock production. These efforts will optimize livestock production under the changing climate scenario. The role of livestock on climate change is primarily due to enteric CH4 emission and those from manure management. Various GHG mitigation strategies include manipulation of rumen microbial ecosystem, plant secondary metabolites, ration balancing, alternate hydrogen sinks, manure management, and modeling to curtail GHG emission. Adapting to climate change and reducing GHG emissions may require significant changes in production technology and farming systems that could affect productivity. Many viable opportunities exist for reducing CH4 emissions from enteric fermentation in ruminant animals and from livestock manure management facilities. To be considered viable, these emission reduction strategies must be consistent with the continued economic viability of the producer and must accommodate cultural factors that affect livestock ownership and management. The direct impacts of climate change on livestock are on its growth, milk production, reproduction, metabolic activity, and disease occurrences. The indirect impacts of climate change on livestock are in reducing water and pasture availability and other feed resources. Amelioration of environmental stress impact on livestock requires multidisciplinary approaches which emphasize animal nutrition, housing, and animal health. It is important to understand the livestock responses to the environment and analyze them, in order to design modifications of nutritional and environmental management, thereby improving animal comfort and performance.

Global Warming: Role of Livestock

This chapter provides an overview of the current state of knowledge concerning global warming with special reference to contribution from livestock resources. Global warming pertains to the effect of natural greenhouse gases (GHGs) such as carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), and halogenated compounds on the environment. These GHGs are generated by humans and human-related activities. Carbon dioxide, CH4, and N2O are the principal sources of radiative forcing (Fifth IPCC Report of 2013). Interestingly, livestock contributes to climate change through emissions of CO2, CH4, and N2O into the atmosphere. Globally, the livestock sector directly and indirectly contributes 18 % (7.1 billion tonnes CO2 equivalent) of GHG emissions. While direct GHG emissions from livestock refer to emissions from enteric fermentations in livestock, urine excretion, and microbial activities in manures, indirect GHG emissions are those not directly derived from livestock activities but from manure applications on farm crops, production of fertilizer for growing crops used for animal feed production, and processing and transportation of refrigerated livestock products. Other indirect emissions include deforestation, desertification, and release of carbons from cultivated soils due to expansion of livestock husbandry. According to FAO’s Global Livestock Environmental Assessment Model (GLEAM), the GHG emission from livestock-related activities was estimated to be around 7.1 gigatonnes CO2-eq. per annum, representing 14.5 % of human-induced emissions. This clearly indicates the significant role for livestock contributions to climate change.

Overview on Adaptation, Mitigation and Amelioration Strategies to Improve Livestock Production Under the Changing Climatic Scenario

Livestock production is thought to be adversely affected by detrimental effects of extreme climatic conditions. Consequently, adaptation, mitigation and amelioration of detrimental effects of extreme climates have played a major role in combating the climatic impact in livestock production. While measures to reduce the growth of greenhouse gas emissions are an important response to the threat of climate change, adaptation to climate change will also form a necessary part of the response. The salient adaptation strategies are developing less sensitive breeds, improving water availability, improving animal health, promoting women empowerment, developing various policy issues, establishing early warning systems and developing suitable capacity building programmes for different stakeholders. Developing adaptation strategies is therefore an important part of ensuring that countries are well prepared to deal with any negative impacts that may occur as a result of climate change. The integration of new technologies into the research and technology transfer systems potentially offers many opportunities to further the development of climate change adaptation strategies. Adapting to climate change and reducing GHG emissions may require significant changes in production technology and farming systems that could affect productivity. Many viable opportunities exist for reducing CH4 emissions from enteric fermentation in ruminant animals and from livestock manure management facilities. To be considered viable, these emission reduction strategies must be consistent with the continued economic viability of the producer and must accommodate cultural factors that affect livestock ownership and management. This chapter also elaborates on ameliorative strategies that should be given consideration to prevent economic losses incurred due to environmental stresses on livestock productivity. Reducing the impact of climatic stresses on livestock production requires multidisciplinary approaches which emphasise animal nutrition, housing and animal health. Therefore, emphasis should be given to all three aspects of adaptation, mitigation and amelioration strategies to sustain livestock production under the changing climate scenario.

Novel insights into the role of cell-free seminal mRNAs on semen quality and cryotolerance of spermatozoa in bulls (Bos taurus)

The aim of the present study was to ascertain the effectiveness of seminal plasma mRNAs as markers to assess the reproductive performance of bulls. Semen samples (33 ejaculates) from 11 bulls were evaluated for sperm kinematic and functional parameters. Total RNA was isolated from cell-free seminal (cfs) using TRIzol LS reagent and the concentration of cfs-RNA was 24.4±2.3µgmL-1 seminal plasma. The cfs-RNA was fragmented to a size of 25-500bp. Of the cfs-mRNAs screened using real time PCR, expression of protamine 1 (PRM1) was positively (P<0.05) associated with the mitochondrial membrane potential of raw semen, whereas expression of Fas Ligand (FASLG) was negatively (P<0.05) associated with sperm velocity, membrane integrity and chromatin distribution in post-thaw semen samples. The percentage of Type A spermatozoa (amplitude of lateral movement of head >2.5μm and straightness >85%) in raw semen was positively (P<0.05) associated with bone morphogenetic protein 2 (BMP2), ubiquitin conjugating enzyme E2D3 (UBE2D3), tumour-associated necrotic factor-associated death domain (TRADD) and caspase-3 (CASP3) expression. Nerve growth factor (NGF) expression was positively (P<0.05) associated with the maintenance of post-thaw functional membrane integrity in spermatozoa and could be used to assess the cryotolerance of bull semen. In conclusion, the expression of cfs mRNAs can be used to assess the reproductive performance of males and to predict the sensitivity of spermatozoa to cryoinjury.

Post-thaw development of in vitro produced buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification

The present study was conducted to examine post-thaw in vitro developmental competence of buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification. In vitro produced embryos were incubated with a medium containing cytochalasin-b (cyto-b) in a CO2 incubator for 40 min for microfilament stabilization and were cryopreserved by a two-step vitrification method at 24℃ in the presence of cyto-b. Initially, the embryos were exposed to 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in a base medium for 4 min. After the initial exposure, the embryos were transferred to a 7 µl drop of 25% EG and 25% DMSO in base medium and 0.3 M sucrose for 45 sec. After warming, the embryos were cultured in vitro for 72 h. The post-thaw in vitro developmental competence of the cyto-b-treated embryos did not differ significantly from those vitrified without cyto-b treatment. The hatching rates of morulae vitrified without cyto-b treatment was significantly lower than the non-vitrified control. However, the hatching rate of cyto-b-treated vitrified morulae did not differ significantly from the non-vitrified control. This study demonstrates that freezing of buffalo embryos by cytoskeletal stabilization and vitrification is a reliable method for long-term preservation.