A. Dhali

Secretion Patterns of Growth Hormone in Growing Captive Mithuns (Bos frontalis)

Abstract A study was conducted in May 2003 to characterize plasma growth hormone (GH) pattern in growing mithuns (Bos frontalis), a rare semi-wild ruminant. Six mithun calves averaging 235 day of age and 124 kg were maintained in semi-intensive system and group-fed once daily. Animals gained at a mean rate of 0.54 kg/day, with individuals ranging from 0.34 to 0.66 kg/day. Blood samples collected at 15-minute intervals starting from 0600h for nine-hour period were assayed for plasma GH. Growth hormone patterns consisted of frequent pulses of varying amplitude. Growth hormone pulses occurred at an average frequency of 0.69/h, the rate did not differ markedly among mithuns nor hour of day. The magnitude of GH secretory pulses varied significantly among mithuns. Growth hormone peaks averaged 95.0 and 45.2 ng/ml in mithuns having the highest and lowest GH peaks, respectively. Peak and mean GH levels were associated positively (r=0.98, P<0.001) and both were associated negatively (r=−0.97 and −0.98, respectively; P<0.01) with rates of gain. Results from the study show that 1) GH peaks occur at frequent intervals throughout the sampling period and 2) alteration in GH levels and patterns are elicited more by pulse amplitude than frequency modulation.

Development and Validation of a Simple, Sensitive, Second Antibody Format Enzyme Immunoassay (EIA) for LH Determination in Mithun (Bos Frontalis) Plasma

Abstract The objective of this study was to develop and validate a simple and highly sensitive enzyme immunoassay (EIA) for LH determination in mithun plasma on microtitreplates using the biotin‐streptavidin amplification system and the second antibody coating technique. Biotin was coupled to LH and used to bridge between streptavidin‐peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 20 µL mithun plasma. The LH standards ranging from 6.25 pg/well/20 µL to 400 pg/well/20 µL were prepared in hormone free plasma collected from a mithun on day 3 post calving. The sensitivity of EIA procedure was 6.25 pg/well LH, which corresponds to 0.31 ng/mL plasma; the 50 percent relative binding sensitivity was seen at 100 pg/well/20 µL. Plasma volumes for the EIA viz. 10 and 20 µL did not influence the shape of standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun plasma LH with bovine LH standards. It showed good parallelism with the bovine standard curve. For the biological validation of the assay, 3 mithuns were used. These were administered 10 µg i.v., with a synthetic analogue of GnRH (Buserelin‐Acetate, Intervet, India) and blood samples were collected at 15 min intervals using indwelling jugular catheter beginning 1 h prior to GnRH injection till 8 h post injection. In all animals, sharp increases in LH concentrations were recorded post GnRH administration, which confirms the biological validation of the EIA. In conclusion, the EIA developed for LH determination in mithun blood plasma is sufficiently reliable, economical, and sensitive enough to estimate LH in all physiological variations in mithun.

Effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa

The effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa was investigated. Semen samples were collected from five healthy mithun bulls through rectal massage method and cryopreserved in liquid nitrogen. The samples were diluted in Tris-egg yolk-glycerol extender, equilibrated for 4 h at 4 °C and loaded into 0.50-ml straws. The straws were then frozen in liquid nitrogen vapour for 10 min and finally plunged into liquid nitrogen for storage. The required amount of glycerol was added into the diluted samples either in a single dose (3%, 4%, 5%, 6% or 7%; added at 37 °C immediately before equilibration) or in split doses (5%, 6% or 7%; the total amount was divided into four equal parts, and a part was added at 37 °C immediately before equilibration, and the remaining parts were added subsequently at 1, 2 and 3 h of equilibration at 4 °C). In the single-dose addition method, following freeze-thawing, greater (p < 0.05) motility (%) and proportion of live spermatozoa with intact acrosome (LSIA, %) in 5% glycerol (40.6 ± 1.7 and 43.4 ± 1.8 respectively) and lesser (p < 0.05) total morphological abnormalities (%) in 5% (14.1 ± 0.8) and 6% (13.7 ± 1.0) glycerol were observed compared to the other glycerol concentrations. In the split-dose addition method, following freeze-thawing, greater (p < 0.05) motility (%) and LSIA proportion (%) were found in 5% (50.2 ± 1.9 and 53.3 ± 1.8 respectively) compared to 6% or 7% glycerol, but the total morphological abnormalities were not different among the glycerol concentrations. In addition, in all the glycerol concentrations, better (p < 0.05) post-freeze-thaw motility and LSIA proportions were observed when glycerol was added in split doses compared to a single dose. In conclusion, Tris-egg yolk extender with 5% glycerol added in split doses was found most suitable for cryopreserving mithun sperm.

Birth of the first mithun ( Bos frontalis) calf through artificial insemination.

The study describes the standardization of a suitable semen cryopreservation protocol for the first time in mithun (Bos frontalis) and birth of the first mithun calf through artificial insemination. The semen samples were collected from adult bulls through the rectal massage method and cryopreserved in liquid nitrogen using tris-egg yolk-glycerol diluent. The diluted semen samples were packaged in 0.50 ml straws and kept at 5°C for 4 h for equilibration. Following the equilibration, the straws were frozen into liquid nitrogen vapour for 10 min and then plunged into liquid nitrogen for storage. It was observed that the progressive motility (%) decreased significantly (P < 0.01) in cryopreserved semen (43.3 ± 4.1) compared with fresh samples (76.6 ± 3.3). The percentages of live spermatozoa (P < 0.01) and spermatozoa with intact acrosome (P < 0.05) also decreased significantly in cryopreserved semen (54.0 ± 3.3 and 64.6 ± 5.3) compared with fresh samples (79.3 ± 2.6 and 85.3 ± 1.8). Simultaneously, the total morphological abnormality (%) was found to be significantly (P < 0.01) higher in cryopreserved samples (15.46 ± 2.68) than in fresh semen (3.85 ± 0.63). A total of three mithun cows were inseminated using the cryopreserved semen. All the cows conceived following insemination and gave birth to healthy calves. The study revealed that mithun semen can be cryopreserved efficiently using tris-egg yolk-glycerol diluent, which can be further used for artificial insemination.

Effect of feeding Lagerstroemia speciosa and conventional fodder based rations on nutrient utilization, ruminal metabolites and body weight gain in mithun (Bos frontalis)

The aim of the present study was to evaluate the effect of feeding green fodder, rice straw and concentrate-based total mixed rations (TMR) on dry matter (DM) intake (DMI), nutrient utilization, rumen fermentation patterns and body weight (BW) gain (BWG) in mithun (Bos frontalis) calves. In a randomized block design, male mithun calves (n = 18, 8-10 months of age, 121 +/- 2 kg BW) were randomly divided into three experimental equal groups (six animals in each group) and fed isonitrogenous TMRs ad libitum for 120 days. The TMR(1) contained 30% Napier grass and 30% rice straw, TMR(2) contained 60% rice straw and TMR(3) contained 30% tree leaves (Lagerstroemia speciosa) and 30% rice straw (DM basis). All the TMRs contained 40% concentrate mixture (DM basis). The results indicated that the BWG, DMI and feed conversion efficiency were significantly (p < 0.01) increased with the inclusion of green fodder in TMRs. The apparent digestibility of DM, crude protein, ether extract, crude fibre and nitrogen free extract were also improved significantly (p < 0.01) with the inclusion of green fodder in TMRs. The higher concentration of total nitrogen and total volatile fatty acid in rumen liquor, but low ruminal pH were evident in animals fed green fodder supplemented TMRs. An increased (p < 0.01) molar proportion of acetic acid was evident in animals fed rice straw-based TMR. In contrast, the molar proportion of propionic and butyric acids were significantly (p < 0.01) higher in animals fed green fodder supplemented TMRs. On the basis of higher DMI and higher daily BWG, it is concluded that Napier grass and L. speciosa tree leaves may be incorporated upto 30% (DM basis) in TMR of growing mithuns for feeding in complete confinement system.

Value addition of corn husks through enzymatic production of xylooligosaccharides

Corn husks are the major wastes of corn industries with meagre economic significance. The present study was planned for value addition of corn husk through extraction of xylan, followed by its enzymatic hydrolysis into xylooligosaccharides, a pentose based prebiotic. Compositional analysis of corn husks revealed neutral detergent fibre 68.87%, acid detergent fibre 31.48%, hemicelluloses 37.39%, cellulose 29.07% and crude protein 2.68%. Irrespective of the extraction conditions, sodium hydroxide was found to be more effective in maximizing the yield of xylan from corn husks than potassium hydroxide (84% vs. 66%). Application of xylanase over the xylan of corn husks resulted into production of xylooligosaccharides with different degree of polymerization namely, xylobiose and xylotriose in addition to xylose monomer. On the basis of response surface model analysis, the maximum yield of xylobiose (1.9 mg/ml) was achieved with the enzymatic hydrolysis conditions of pH 5.8, temperature 44°C, enzyme dose 5.7U/ml and hydrolysis time of 17.5h. Therefore, the corn husks could be used as raw material for xylan extraction vis a vis its translation into prebiotic xylooligosaccharides.

Assessment of Fecal Microflora Changes in Pigs Supplemented with Herbal Residue and Prebiotic

Antibiotic usage in animals as a growth promoter is considered as public health issue due to its negative impact on consumer health and environment. The present study aimed to evaluate effectiveness of herbal residue (ginger, Zingiber officinale, dried rhizome powder) and prebiotic (inulin) as an alternative to antibiotics by comparing fecal microflora composition using terminal restriction fragment length polymorphism. The grower pigs were offered feed containing antibiotic (tetracycline), ginger and inulin separately and un-supplemented group served as control. The study revealed significant changes in the microbial abundance based on operational taxonomic units (OTUs) among the groups. Presumptive identification of organisms was established based on the fragment length of OTUs generated with three restriction enzymes (MspI, Sau3AI and BsuRI). The abundance of OTUs representing Bacteroides intestinalis, Eubacterium oxidoreducens, Selonomonas sp., Methylobacterium sp. and Denitrobacter sp. was found significantly greater in inulin supplemented pigs. Similarly, the abundance of OTUs representing Bacteroides intestinalis, Selonomonas sp., and Phascolarcobacterium faecium was found significantly greater in ginger supplemented pigs. In contrast, the abundance of OTUs representing pathogenic microorganisms Atopostipes suicloacalis and Bartonella quintana str. Toulouse was significantly reduced in ginger and inulin supplemented pigs. The OTUs were found to be clustered under two major phylotypes; ginger-inulin and control-tetracycline. Additionally, the abundance of OTUs was similar in ginger and inulin supplemented pigs. The results suggest the potential of ginger and prebioticsto replace antibiotics in the diet of grower pig.

Effect of method and time of first colostrum feeding on serum immunoglobulin concentration, health status and body weight gain in mithun (Bos frontalis) calves

The effect of method and time of first colostrum feeding on the concentration of serum immunoglobulin (Ig) was evaluated in mithun (Bos frontalis) calves. The hypotheses were that the variable method and time of first colostrum feeding might affect the level of serum Ig and in turn the growth performance and health status of the claves during the early age. The newborn calves were randomly allotted to one of the four experimental groups - G-1: allowed to suckle the dam at own choice, G-2: separated immediately after birth and allowed to suckle the dam first at 6 h and then at own choice, G-3: bottle fed ad libitum colostrum of its own dam first at 6 h and then at 6-h intervals until 24 h, G-4: bottle fed ad libitum colostrum of its own dam within 1 h, at 6 h and then at 6-h intervals until 24 h. The concentrations of IgG, IgM, and IgA were lowest (p < 0.01) at birth and increased following colostrum feeding irrespective of the experimental group. Highest concentrations of all the Ig classes were observed at 12-24 h after birth. The concentrations then transiently decreased from day 7 to 14, and then steadily increased after day 28. The concentrations of IgG (p < 0.01) and IgA (p < 0.05) were higher in G-1 in relation to the other groups during the first week after birth. Similarly, higher concentration of IgA (p < 0.05) was found in G-1 in relation to the other groups during the rest of the experimental period. The apparent absorption efficiency of colostral IgG was higher (p < 0.05) in G-4 in relation to G-3. Growth rate and health status were not influenced by the method and time of first colostrum feeding. In conclusion, a 6-h delay in the first colostrum feeding reduced the level of serum Ig noticeably.

Methane mitigation potential of phyto-sources from Northeast India and their effect on rumen fermentation characteristics and protozoa in vitro

Aim: The aim of the study was to explore the anti-methanogenic potential of phyto-sources from Northeast region of the country and assess the effect on rumen fermentation characteristics and protozoa for their likely inclusion in animal diet to reduce methane emission. Materials and Methods: Twenty phyto-sources were collected from Northeast state, Assam, during March to April 2014. Phyto-sources were analyzed for their tannin content followed by screening for methane mitigation potential using in vitro system. The effect of tannin on methane production and other fermentation parameters was confirmed by attenuating the effect of tannin with polyethylene glycol (PEG)-6000 addition. About 200 mg dried phyto-source samples were incubated for 24 h in vitro, and volume of gas produced was recorded. The gas sample was analyzed on gas chromatograph for the proportion of methane in the sample. The effect of phyto-sources on rumen fermentation characteristics and protozoal population was determined using standard methodologies. Results: Results from studies demonstrated that Litchi chinensis, Melastoma malabathricum, Lagerstroemia speciosa, Terminalia chebula, and Syzygium cumini produced comparatively less methane, while Christella parasitica, Leucas linifolia, Citrus grandis, and Aquilaria malaccensis produced relatively more methane during in vitro incubation. An increase (p<0.05) in gas and methane production from the phyto-sources was observed when incubated with PEG-6000. Entodinimorphs were prominent ciliates irrespective of the phyto-sources, while holotrichs represented only small fraction of protozoa. An increase (p<0.05) in total protozoa, entodinimorphs, and holotrichs was noted when PEG-6000 added to the basal substrate. Our study confirmed variable impact of phyto-sources on total volatile fatty acid production and ammonia-N. Conclusion: It may be concluded that L. chinensis, M. malabathricum, L. speciosa, S. cumini, and T. chebula are having potent methane suppressing properties as observed in vitro in 24 h. These leaves could be supplemented in the animal diet for reducing methane emission; however, in vivo trials are warranted to confirm the methane inhibitory action and optimize the level of supplementation.

Development and validation of a sensitive radioimmunoassay procedure for estimating FSH in mithun (Bos frontalis) plasma.

Abstract The present study was designed to develop and validate a simple and sensitive radioimmunoassay (RIA) procedure to estimate FSH in mithun (Bos frontalis) plasma. The assay was carried out in 100 µL of mithun plasma. The bovine FSH standards (10 to 5000 pg/100 µL/tube) in hormone‐free plasma were used in the assay. The sensitivity of the assay was 20 pg/100 µL/tube, which corresponded to 0.20 ng/mL plasma. The 50% relative binding sensitivity of the assay was 80 pg/100 µL/tube, which corresponded to 0.80 ng/mL plasma. The intra‐ and inter‐assay coefficients of variation were 4.6% and 12.4%, respectively. The biological validation of the assay was carried out in plasma samples that were collected during different stages of the estrous cycle. In the entire estrous cycle, plasma FSH concentration (p<0.01) attained two peaks (on day 3 to 4 before estrus 5.1±0.3 ng/mL and on the day of estrus 6.9±0.2 ng/mL). FSH concentration remained at basal level (1.3±0.1 to 1.6±0.2 ng/mL) during day 4 to 16 of the estrous cycle. The concentration of plasma FSH was found to be significantly (p<0.05) higher (4.9±0.3 to 6.8±0.5 ng/mL) until 48 h following the estrus onset. In conclusion, the RIA procedure that was developed in the current study is sufficiently reliable and sensitive to estimate different physiological levels of FSH in mithun plasma.