Muscle transcriptome signature and gene regulatory network analysis in two divergent lines of a hilly bovine species Mithun (Bos frontalis).

A massive bovine, Bos frontalis, also known as Mithun or Gayal, found at higher altitude is very promising meat and milk animal. For candidate gene and marker discovery, RNA-seq data was generated from longissimus dorsi muscle tissues with Illumina-HiSeq. Such markers can be used in future for genetic gain of traits like feed conversion efficiency (FCE) and average daily gain (ADG). Analysis revealed 297differentially expressed genes (DEGs) having 173 up and 124 down-regulated unigenes. Extensive conservation was found in genic region while comparing with Bos taurus. Analysis revealed 57 pathways having 112 enzymes, 72 transcriptional factors and cofactors, 212 miRNAs regulating 71 DEGs, 25,855 SSRs, mithun-specific 104,822 variants and 7288 indels, gene regulatory network (GRN) having 24 hub-genes and transcriptional factors regulating cell proliferation, immune tolerance and myogenesis. This is first report of muscle transcriptome depicting candidate genes with GRN controlling FCE and ADG. Reported putative molecular markers, candidate genes and hub proteins can be valuable genomic resources for association studies in genetic improvement programme.

Whole genome sequence and de novo assembly revealed genomic architecture of Indian Mithun (Bos frontalis).

BACKGROUND: Mithun (Bos frontalis), also called gayal, is an endangered bovine species, under the tribe bovini with 2n = 58 XX chromosome complements and reared under the tropical rain forests region of India, China, Myanmar, Bhutan and Bangladesh. However, the origin of this species is still disputed and information on its genomic architecture is scanty so far. We trust that availability of its whole genome sequence data and assembly will greatly solve this problem and help to generate many information including phylogenetic status of mithun. Recently, the first genome assembly of gayal, mithun of Chinese origin, was published. However, an improved reference genome assembly would still benefit in understanding genetic variation in mithun populations reared under diverse geographical locations and for building a superior consensus assembly. We, therefore, performed deep sequencing of the genome of an adult female mithun from India, assembled and annotated its genome and performed extensive bioinformatic analyses to produce a superior de novo genome assembly of mithun. RESULTS: We generated ≈300 Gigabyte (Gb) raw reads from whole-genome deep sequencing platforms and assembled the sequence data using a hybrid assembly strategy to create a high quality de novo assembly of mithun with 96% recovered as per BUSCO analysis. The final genome assembly has a total length of 3.0 Gb, contains 5,015 scaffolds with an N50 value of 1 Mb. Repeat sequences constitute around 43.66% of the assembly. The genomic alignments between mithun to cattle showed that their genomes, as expected, are highly conserved. Gene annotation identified 28,044 protein-coding genes presented in mithun genome. The gene orthologous groups of mithun showed a high degree of similarity in comparison with other species, while fewer mithun specific coding sequences were found compared to those in cattle. CONCLUSION: Here we presented the first de novo draft genome assembly of Indian mithun having better coverage, less fragmented, better annotated, and constitutes a reasonably complete assembly compared to the previously published gayal genome. This comprehensive assembly unravelled the genomic architecture of mithun to a great extent and will provide a reference genome assembly to research community to elucidate the evolutionary history of mithun across its distinct geographical locations.

Intake, digestibility and nitrogen balance in Mithun (Bos frontalis) offered urea-treated paddy straw based feed blocks.

The aim of the study was to determine the effect of feeding feed blocks containing varying proportion of urea-treated paddy straw (UTPS) on dry matter (DM) intake (DMI), nutrient utilization and N balance in Mithun. For the purpose, four adult male Mithun (279.5 ± 8.2 kg) were selected and offered four experimental rations viz. R(1) (Napier fodder + concentrate at 60:40), R(2) (UTPS + concentrate at 50:50), R(3) (UTPS + concentrate at 60:40) and R(4) (UTPS + concentrate at 70:30) in 4 × 4 Latin square design. The DMI % of body weight was 2.59, 2.96, 2.85 and 2.77 and the DMI g kg(-1) W(0.75) was 107, 123, 118 and 115 in Mithun fed R(1), R(2), R(3) and R(4), respectively. The mean DMI was (P < 0.01) higher in animals fed R(2) and R(3) than R(1) and R(4), whereas the water intake was (P < 0.01) higher in Mithun fed R(2), R(3) and R(4) than R(1). The digestibility of DM, organic matter, crude protein, crude fibre, neutral detergent fibre and cellulose were (P < 0.05) higher in animals fed R(2), R(3) and R(4) than R(1). A positive N balance was observed in all the experimental animals, with higher (P < 0.05) values among the animals offered R(2), R(3) and R(4) than R(1). The digestible crude protein and total digestible nutrient intakes were higher (P < 0.05) in Mithun fed R(2) and R(3) than R(1) and R(4). It is concluded that the UTPS can be incorporated up to 70% to formulate the complete feed/feed block and can be used for feeding of Mithun under complete confinement system.

High-density Genotyping reveals Genomic Characterization, Population Structure and Genetic Diversity of Indian Mithun (Bos frontalis).

The current study aimed at genomic characterization and improved understanding of genetic diversity of two Indian mithun populations (both farm, 48 animals and field, 24 animals) using genome wide genotype data generated with Illumina BovineHD BeadChip. Eight additional populations of taurine cattle (Holstein and NDama), indicine cattle (Gir) and other evolutionarily closely related species (Bali cattle, Yak, Bison, Gaur and wild buffalo) were also included in this analysis (N = 137) for comparative purposes. Our results show that the genetic background of mithun populations was uniform with few possible signs of indicine admixture. In general, observed and expected heterozygosities were quite similar in these two populations. We also observed increased frequencies of small-sized runs of homozygosity (ROH) in the farm population compared to field mithuns. On the other hand, longer ROH were more frequent in field mithuns, which suggests recent founder effects and subsequent genetic drift due to close breeding in farmer herds. This represents the first study providing genetic evidence about the population structure and genomic diversity of Indian mithun. The information generated will be utilized for devising suitable breeding and conservation programme for mithun, an endangered bovine species in India.

Highly sensitive second antibody format enzymeimmunoassay for determination of 13,14-dihydro-15-keto-PGF2alpha in blood plasma of mithun (Bos frontalis).

As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM) in blood plasma of mithun (Bos frontalis; bovine) on microtitreplates using second antibody coating technique and PGFM-horseradish peroxidase as a label has been developed. The wells of the microtitreplate were coated with affinity-purified goat IgG (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20microl plasma. The PGFM standard curve, with doses ranging from 0.1 to 50pg/well was linear. The sensitivity of the assay was 5pg/ml. PGFM standard curve in buffer showed parallelism with serially diluted mithun plasma containing high endogenous PGFM. Plasma PGFM concentrations estimated by using the developed EIA and commercially available PGFM EIA kit in the same samples were significantly correlated (r=0.98) and showed linearity. Intra- and inter-assay coefficients of variation were below 7%. Recovery of known concentrations of added PGFM in charcoal stripped plasma was linear (r=0.99). The developed EIA was further validated biologically by estimating PGFM in cyclic cows for the entire estrous cycle and in peri-parturient cows beginning day 7 prior to calving till day 30 post-calving; the concentrations were along with the expected lines as reported in bovine. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of PGFM directly in bovine plasma.

Development of a biotin-streptavidin amplified enzyme immunoassay for oxytocin and its application during milk ejection and the reproductive cycle in the mithun (Bos frontalis).

Oxytocin is a key hormone involved in milk ejection. It plays a key role in regulation of reproductive cyclicity in female mammals by taking part in the process of luteolysis. Determination of oxytocin is, therefore, important for studying the control of its secretion and its role in reproduction of the mithun. A simple and sufficiently sensitive enzyme immunoassay (EIA) for oxytocin determination in mithun plasma using the biotin-streptavidin amplification system and second antibody coating technique was therefore developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in a competitive assay. The EIA was conducted directly in 200 microl of unknown mithun plasma. Standards prepared in hormone-free plasma were used. The lowest detection limit was 0.5 pg/ml plasma. Plasma volumes for the EIA (50, 100, and 200 microl) did not influence the shape of standard curve, even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare endogenous mithun oxytocin with a bovine oxytocin standard. The former showed good parallelism with the bovine standard curve. For biological validation of the assay, plasma oxytocin was measured in the blood samples collected before, during, and after milking in three mithun cows and in six non-lactating cyclic mithuns during the entire estrous cycle. A sharp release of oxytocin shortly after udder stimulation was observed. A high level of oxytocin was maintained during milking, falling sharply thereafter. The mean plasma oxytocin concentration was different on different days of the estrous cycle (P < 0.001). Two peaks of oxytocin were recorded, one at day 6 and another at day 18 of the estrous cycle. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine plasma oxytocin levels in mithuns. Apart from being non-radioactive, the EIA procedure described here also utilizes a highly stable biotinalyted hormone which has a shelf life of several years, unlike the short shelf life of iodinated tracer used in RIA procedures.

Behavioral estrous signs can predict the time of ovulation in mithun (Bos frontalis).

The objective of this study was to investigate the relationship of different behavioral estrous signs and time of ovulation to identify if behavioral estrous sign(s) can be used as predictor of time of ovulation in mithuns. Data were collected for 54 ovulations from 16 mithuns. The animals were monitored for onset of estrus by observing different behavioral estrous signs at 2 h interval and bull parading thrice a day for 30 min and were further confirmed by plasma progesterone profile. All animals were also observed for any of the estrous signs at every 2 h interval for 30 min and mounting behavior was studied by bull parading at every 2 h for 30 min after onset of estrus. Time of ovulation was detected by rectal palpation at 2 h interval from onset of estrus till ovulation. Behavioral signs of estrus was more intense in primiparous than multiparous mithuns. Ovulation occurred at 26.1+/-1.1 h (ranging between 20 and 31 h) after the onset of estrus. As the method used to determine the onset of estrus is time consuming, labor intensive and no device is yet available to detect onset of estrus automatically, so this cannot be used practically as a predictor of time of ovulation. The mithun cow at estrus to be mounted by bull was recorded in all cases (100%). Ovulation occurred 23.5+/-1.5 h (ranging between 19 and 27 h) after first mounting. Although promising, mounting cannot be assessed automatically, which limits its practical use as a predictor of ovulation. Standing heat was recorded in 98.1% of total estrus studied in mithun cows and ovulation occurred 21.8+/-1.3 h (ranging between 19 and 25 h) after first observed standing heat. Standing heat can be detected automatically using mounting detectors. Hence, standing heat can be used practically as ovulation predictor in mithuns. In conclusion, cow to be mounted by mithun bull is the best predictor of ovulation, but non-availability of devices to detect it automatically restricts its practical application. Standing heat that recorded 98.1% estrus cases in mithun cows, can also be detected automatically using mounting detector, therefore be used widely as an ovulation predictor in field condition for mithun cows.

Diurnal changes in blood metabolites and their relation to plasma growth hormone and time of feeding in mithun heifers (Bos frontalis).

The aim of the present study was to investigate what, if any, diurnal changes occur in blood metabolites in relation to plasma growth hormone (GH) and feeding time among mithun (Bos frontalis), a semi-wild ruminant. Blood samples were collected at hourly intervals during a 24 h span from 6 mithun heifers (averaging 2.5 yr of age and averaging 230 kg in weight) that were fed twice a day at 11:00 and 16:00 h. Samples were assayed for plasma GH and blood metabolites, non-esterified fatty acids (NEFA), glucose, and alpha-amino nitrogen. The total sampling period was divided into a 1) postprandial (after meal) period (period I: 11:00 to 21:00 h) and 2) interprandial period (period II: 22:00 to 10:00 h) and also into night (20:00 to 05:00 h) and day (06:00 to 10:00 h) periods for statistical analysis. Plasma glucose and alpha-amino nitrogen levels increased (p<0.01), and plasma NEFA and GH decreased (p<0.01) after each meal. No diurnal rhythmicity was detected in plasma glucose or alpha-amino nitrogen levels. Interestingly, plasma NEFA and GH levels were higher (p<0.01) during the interprandial (period II) and night periods, indicating an energy deficit that occurred progressively during the interprandial period of nocturnal feed deprivation. In twice-daily-fed mithuns we conclude that: 1) plasma metabolites and GH exhibited a definite pattern of change with time of feeding; 2) concentrations of plasma NEFA were higher nocturnally due to an energy deficit and that GH levels were higher during the interprandial period after the second meal; 3) the interprandial period after the second feeding may be considered to constitute a short-term food deprivation; 4) the longer interprandial period of 19 h in this study between the second and subsequent morning meal may be changed into equally divided feedings to minimize the short-term energy deficit; and 5) blood sampling for blood metabolites in mithuns should be conducted at a fixed time of day with special emphasis on time of feeding.

Secretion patterns of luteinizing hormone in growing mithuns (Bos frontalis).

To characterize the luteinizing hormone (LH) secretion patterns in growing mithun (Bos frontalis), a semi-wild ruminant, six female mithuns (1 year old; BW: 145.5 kg) were maintained in a semi-intensive system. Plasma progesterone (P(4)) level was measured in twice-a-week samples collected for six weeks to assess ovarian status. This was followed by a frequent sampling period. Blood samples collected at 15 min intervals for 9 h were assayed for plasma LH. Luteinizing hormone patterns consisted of pulses of varying amplitudes. Luteinizing hormone pulses occurred at an average rate of 0.54/h ( approximately 5 pulses/9 h). The rate did not differ among mithuns. The mean plasma LH levels was correlated with body weight (r=0.82; p<0.05) and pulse amplitude (r=0.87; p<0.01). Neither the LH amplitude nor the frequency was affected by time (p>0.05). The mean plasma P(4) concentration was 0.37 ng/ml. In conclusion, we demonstrated a pulsatile nature of LH secretion in growing mithuns. In addition, the mean plasma LH level and LH amplitude were positively correlated with body weight. It appears that in contrast to cattle, five LH pulses per nine hours recorded in mithuns were not an indication of approaching puberty.

Secretion patterns of growth hormone in growing captive mithuns (Bos frontalis).

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.

Characterization of Mithun (Bos frontalis) ejaculates and fertility of cryopreserved sperm.

Technologies for conservation and propagation of genetic resources in the Mithun (Bos frontalis), a rare semi-wild bovine species of Southeast Asia. Successful cryopreservation of Mithun semen would provide a potential vehicle to address above issue. To date, information on characteristics of Mithun ejaculates is not available and there are no reports of birth of live offspring using cryopreserved Mithun semen collected using AV method. A study was therefore conducted to (i) characterize the Mithun ejaculate, (ii) investigate the effectiveness of Mithun sperm cryopreservation, and (iii) determine whether artificial insemination using frozen-thawed Mithun sperm can result in live offspring. Semen samples collected from eight fertile Mithun bulls were evaluated for colour, consistency, volume, concentration, mass activity and progressive motility. The freshly ejaculated sperm were also evaluated for morphological abnormalities, live sperm counts, acrosome integrity, membrane stability (hypo-osmotic swelling test; HOST) and DNA integrity. Semen samples of good quality were cryopreserved in liquid nitrogen using Tris-egg yolk-glycerol diluent. Post-thaw quality of the cryopreserved sperm in terms of progressive motility, morphological abnormalities, live sperm counts, acrosome integrity, membrane stability and DNA integrity were assessed. In addition, 16 Mithun cows at estrus were inseminated with frozen-thawed Mithun sperm. Following cryopreservation, the percentage of progressive motility (fresh versus frozen-thawed), live sperm counts, morphological abnormalities, acrosome integrity, membrane stability and DNA integrity were found to decrease (P<0.01) with a motility recovery rate of 74+/-9%. Mithun cows inseminated with cryopreserved sperm result 75% conception rate and all the conceived cows maintained full-term pregnancy with delivery of live calves.

Electrocardiogram of Clinically Healthy Mithun (Bos frontalis): Variation among Strains.

A study was conducted to establish the normal electrocardiogram in four different genetic strains of mithun (Bos frontalis). Electrocardiography, cardiac electrical axis, heart rate, rectal temperature and respiration rate were recorded in a total of 32 adult male mithun of four strains (n = 8 each). It was found that the respiration and heart rates were higher (P < .05) in Manipur than other three strains. Amplitude (P < .05) and duration of P wave and QRS complex differed (P < .01) among the strains. Mizoram strain had the highest amplitude and duration of P wave and QRS complex. On the other hand, higher (P < .05) amplitude and duration of T wave were recorded in Arunachalee and Mizoram strains. The mean electrical axis of QRS complex that were recorded for Arunachalee and Manipur strains were similar to that reported for other bovine species; whereas the electrical axis of QRS for Nagamese and Mizoram strains were more close to feline and caprine species, respectively. In conclusion, electrocardiogram of mithun revealed that the amplitude and duration of P wave, QRS complex and T wave were different among four different genetic strains of mithun and the electrical axis of QRS complex for Nagamese and Mizoram mithuns are dissimilar to bovine species.

Relationship of plasma estradiol-17beta, total estrogen, and progesterone to estrus behavior in mithun (Bos frontalis) cows.

The objectives of this study were (1) to establish the characteristics of estrus behavior in mithun cows (n = 12) and (2) to determine the relationships between this behavior and the plasma concentrations of estradiol-17beta (E2), total estrogen, and progesterone. Estrus was detected by visual observations of estrus signs, per recta examination of genitalia and bull parading thrice a day for three consecutive cycles. Among the behavioral signs of estrus, the cow to be mounted by bull (100%) was the best indicator of estrus followed by standing to be mounted (92%). Per rectum examination of genital organs revealed relaxed and open os externa of cervix, turgid uterus, and ovaries having palpable follicles in all animals. The mean (+/-SEM) length of estrus cycle and duration of estrus were recorded to be 21.8 +/- 0.69 days and 12.6 +/- 1.34 h, respectively. Endocrine profiles during the peri-estrus period showed that the mean highest peak concentrations of E2 (27.29 +/- 0.79 pg/ml) and total estrogen (45.69 +/- 2.32 pg/ml) occurred at -3.90 +/- 2.27 and -3.89 +/- 2.26 h prior to the onset of estrus, respectively. Plasma progesterone concentration was basal (0.14 +/- 0.001 ng/ml) during the peri-estrus period. Plasma E2 and total estrogen were found to increase from 6 days before estrus to reach a peak level on the day of estrus and decline thereafter to basal level on day 3 of the cycle. The plasma progesterone concentration was the lowest on the day of estrus showing gradual increase to register a peak level on day 15 of the cycle. Estrus behavior was found to be positively correlated with the maximum peak concentration of E2 (r = 0.89; P < 0.0001) and total estrogen (r = 0.66; P = 0.019) during the peri-estrus period. The mean total estrogen concentration during the peri-estrus period was significantly correlated with estrus behavior (r = 0.60; P = 0.04). The correlations between the estrus behavior and E2:progesterone ratios at 6 days before the onset of estrus (r = 0.92) and on the day of estrus (r = 0.95) was significant. The total estrogen:progesterone ratios at 6 days before the onset of estrus and on the day of estrus were also positively correlated with the estrus behavior (r = 0.86 and 0.88). In conclusion, our results suggest that the maximum peak concentration of E2 and total estrogen and mean level of total estrogen during the peri-estrus period and the E2:progesterone and total estrogen:progesterone ratios on 6 days before the onset of estrus and on the day of estrus are the important factors contributing the behavioral manifestation of estrus in mithun cows.

Exogenous GH-releasing hormone increases GH and LH secretion in growing mithuns (Bos frontalis).

To investigate the effects of growth hormone-releasing hormone (GHRH) administration on the patterns of GH and LH secretion in growing female mithuns, 12 mithuns within the age group of 10-12 months of age were divided into two groups (treatment and control groups) of six each in such a way that average body weight between the groups did not differ significantly (P>0.05). Both the groups were administered i.v. either with synthetic bGHRH [bGHRH (1-44)-NH2] at 10 microg/100 kg body weight (treatment group) or equal volume of normal saline (control group). Blood samples collected prior to and after GHRH challenge at -60, -45, -30, -15, -10, -5 min and 5, 10, 15, 30 min, and thereafter, at an interval of 15 min up to 8 h post-injection were assayed for plasma GH and LH. Plasma progesterone was estimated in twice-a-week samples collected for six consecutive weeks preceding GHRH challenge to assess whether either group has begun ovarian cyclicity. Body weight of all animals was recorded once in a week during the period. A peak of GH was registered in all animals within 5-25 min post-GHRH administration with a mean peak of 443.5+/-25.32 ng/ml at 15 min post-administration, which was much higher than in any other bovines reported following GHRH challenge. The patterns of LH secretion were pulsatile in nature in both the groups. Interestingly, the hormone concentrations exhibited higher pulsatility with greater amplitude after GHRH challenge in GHRH-treated than in control mithuns. The GHRH-treated mithuns averaged 0.44 pulses/h (4 pulses/9 h) and the rate was 0.20/h (2 pulses/9 h) in controls. The rate of pulse frequency and amplitude differed significantly with time of sampling. The mean plasma LH levels after GHRH administration were significantly higher in treatment group than those recorded in control mithuns. The mean plasma progesterone was similar (P>0.05) in both the groups and no animal from either group had begun ovarian cycle. In conclusion, exogenous GHRH significantly increases plasma GH and also LH pulse frequency and amplitude with higher mean post-GHRH LH levels in growing mithuns suggesting thereby its possible use for enhancement of maturity process in this unique meat animal.

Determination of effective dosage of GH-releasing factor for blood GH responses in mithun (Bos frontalis).

A study was undertaken to determine the effective dosage of GH-releasing hormone (GRF) required to produce blood GH response in mithun (Bos frontalis), a semi-wild ruminant species. For the purpose, 12 mithuns averaging 11.5 months of age and 146 kg body weight (BW) were randomly assigned to receive GRF (n = 12), administered at 0 (normal saline), 5, 10 and 20 mug per 100 kg BW. Blood samples were collected prior to and after GRF administration at -60, -45, -30, -15, -10, -5, 0 min and 5, 10, 15, 30 and thereafter, at 15-min interval up to 8 h post-GRF were assayed for plasma GH. For all the dosages, the pre-treatment GH concentrations and corresponding area under GH response curve (AUC) were similar (p > 0.05). The post-GRF plasma GH responses to different dosages of GRF viz. 5, 10 and 20 mug per 100 kg BW and corresponding AUCs were higher (p < 0.05) than those recorded in normal saline-treated controls. The GH responses to 10 and 20 mug GRF per 100 kg BW and corresponding AUCs were higher (p < 0.05) than those registered in mithuns administered with 5 mug GRF per 100 kg BW. Interestingly, post-GRF concentration of plasma GH and AUCs were not different for 10 and 20 mug GRF per 100 kg BW dosages. In all animals treated with GRF, a peak of GH was registered within 10 to 20 min post-GRF. Following 5 mug GRF per 100 kg BW, GH concentrations were maintained at higher level for 90 min post-GRF and thereafter became similar to that of controls and it was 435 min for 10 and 20 mug GRF per 100 kg BW dosages. In conclusion, our results suggest that 10 mug GRF per 100 kg BW is the dosage, which can be used for augmentation of mithun production.

Relationship between plasma growth hormone concentrations and temperament in mithuns (Bos frontalis).

The aim of the present study was to verify whether or not plasma growth hormone (GH) concentrations are correlated with temperament in mithuns (Bos frontalis), a semiwild ruminant. Therefore, a total of 69 female mithuns from four different strains, viz., Arunachal, Nagaland, Mizoram, and Manipur, were divided into six age groups (Group I, 0-6 months; Group II, >6-12 months; Group III, >1-2 years; Group IV, >2-2.5 years; Group V, >2.5-3.0 years; Group VI, >3.0 years). Blood samples were collected weekly for 6 consecutive weeks and assayed for plasma GH. Temperament was scored on a 6-point scale, 6 were being very aggressive and 1 docile. Body weights of all animals were recorded once a week for 6 consecutive weeks GH concentrations and temperament scores were found to differ significantly between groups. Strain had significant effects on blood GH levels and temperament. Blood GH concentrations and temperament of Manipur mithuns were significantly higher than those of the other three strains within each group, for all groups. Across groups III to VI, blood GH levels and temperament among Nagaland, Mizoram, and Arunachal mithuns did not differ. Overall, the strain with the highest blood GH concentrations also had highest temperament scores. The Manipur strain had the highest blood GH levels and exhibited the most aggression (r = 0.95), and Arunachal mithuns, the lowest (r = 0.93). Temperament scores tended to decrease with increasing age for all four strains. Coefficients of correlation between blood GH and temperament among strains within each group, for all groups, were found to be significant. The highly positive correlation (r = 0.94) between blood GH concentrations and temperament for all animals, regardless of age and strain differences, clearly indicates the relationship between blood GH and temperament in mithuns. In conclusion, our results suggest that peripheral blood GH levels can influence temperament in mithuns.

Twenty-four-hour secretion patterns of luteinizing hormone in mithuns (Bos frontalis).

A 24 h secretion pattern of luteinizing hormone (LH) was not available in mithun (Bos frontalis), a semi-wild ruminant. To characterize the 24 h LH profiles, six female mithun calves (age 7.8 +/- 0.5 months and 102.5 +/- 5.6 kg; group I) and six female mithuns averaging 25.4 months of age and 240 kg (group II) were selected from the National Research Centre on Mithun farm and were maintained in semi-intensive systems. Blood samples collected from all the animals at 30 min intervals for 24h were assayed for plasma LH. Plasma progesterone was also estimated in twice-a-week samples collected for 6-week period preceding each 24h sampling to assess whether any animal had begun ovarian cyclicity. The body weights of all animals were also recorded weekly during the 6-week period. LH patterns consisted of frequent pulses of varying amplitude. Luteinizing hormone pulses occurred at an average frequency of 0.28/h ( approximately 7 pulses/24 h) and 0.15/h ( approximately 3.5 pulses/24 h) for mithuns of groups II and I, respectively, the rate did not differ markedly among mithuns within each group but was significantly different between the groups. Similarly, the magnitude of LH secretory pulses did not vary among mithuns within the group but was significantly higher in group II than in group I animals. In group II, the LH peaks averaged 1.59 and 1.00 ng/ml in mithun having the highest and lowest LH peaks, respectively and the corresponding values for group I mithuns were 0.66 and 0.51ng/ml. Mithun with higher peak LH levels also had higher mean LH concentrations (P<0.05). The mithuns of group II had significantly higher plasma progesterone concentration (0.89 +/- 0.02 ng/ml) than those recorded in group I mithuns (0.26 +/- 0.01 ng/ml). Plasma progesterone profiles suggested that no animal reached puberty. In conclusion, there was higher LH secretion with higher pulsatility and greater amplitude in group II mithuns than exhibited in mithuns of group I and the prepubertal mithuns of group II were in approaching puberty, which were also indicated by their plasma progesterone profiles, critical body weight and age required to attain puberty, in addition to higher pulsatility of LH secretion.

Development and validation of a sensitive radioimmunoassay for progesterone estimation in unextracted mithun (Bos frontalis) plasma.

The objective of the study was to develop and validate a simple, reliable, and highly sensitive radioimmunoassay (RIA) for progesterone determination in mithun (Bos frontalis) plasma. The RIA was carried out in 20 microL unextracted mithun plasma. The progesterone standards ranging from 2 to 500pg/20 microL/tube were prepared in charcoal-treated hormone-free plasma. The sensitivity of the RIA procedure was 2 pg progesterone/20 microL/tube, which corresponds to 0.1 ng/mL; the 50 percent relative binding sensitivity was seen at 32pg/20 microL/tube. Plasma volumes for the RIA, viz. 10 and 20 microL, did not influence the shape of standard curve, even though a slight drop in the counts was seen with higher plasma volumes. For the biological validation of the assay, three cyclic, three in early pregnancy, and two in late pregnancy mithuns were used. Blood samples collected at weekly intervals for 42 days, from all animals, were assayed for plasma progesterone. The peak level of progesterone was registered at day 14 (day 21 of sampling) of the estrous cycle and the lowest at estrus; the progesterone concentrations increased and decreased gradually as sampling time advanced, in early and late pregnancy, respectively, which confirm the biological validation of the RIA. The RIA avoids the troublesome and laborious plasma extraction procedures. In conclusion, the RIA developed for progesterone determination in mithun blood plasma is sufficiently reliable, simple, and sensitive enough to estimate progesterone in all physiological variations in mithun.

Development and validation of a simple, sensitive, second antibody format enzyme immunoassay (EIA) for LH determination in mithun (Bos frontalis) plasma.

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 microL mithun plasma. The LH standards ranging from 6.25 pg/well/20 microL to 400 pg/well/20 microL 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 microL. Plasma volumes for the EIA viz. 10 and 20 microL 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 microg 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.