Vijay Kumar Saxena

Sequence analysis and identification of new variations in the coding sequence of melatonin receptor gene (MTNR1A) of Indian Chokla sheep breed

Melatonin receptor 1A gene is the prime receptor mediating the effect of melatonin at the neuroendocrine level for control of seasonal reproduction in sheep. The aims of this study were to examine the polymorphism pattern of coding sequence of MTNR1A gene in Chokla sheep, a breed of Indian arid tract and to identify new variations in relation to its aseasonal status. Genomic DNAs of 101 Chokla sheep were collected and an 824 bp coding sequence of Exon II was amplified. RFLP was performed with enzyme RsaI and MnlI to assess the presence of polymorphism at position C606T and G612A, respectively. Genotyping revealed significantly higher frequency of M and R alleles than m and r alleles. RR and MM were found to be dominantly present in the group of studied population. Cloning and sequencing of Exon II followed by mutation/polymorphism analysis revealed ten mutations of which three were non-synonymous mutations (G706A, C893A, G931C). G706A leads to substitution of valine by isoleucine Val125I (U14109) in the fifth transmembrane domain. C893A leads to substitution of alanine by aspartic acid in the third extracellular loop. G931C mutation brings about substitution of amino acid alanine by proline in the seventh transmembrane helix, can affect the conformational stability of the molecule. Polyphen-2 analysis revealed that the polymorphism at position 931 is potentially damaging while the mutations at positions 706 and 893 were benign. It is concluded that G931C mutation of MTNR 1A gene, may explain, in part, the importance of melatonin structure integrity in influencing seasonality in sheep.

Assessment of Genetic Variability in the Coding Sequence of Melatonin Receptor Gene (MTNR1A) in Tropical Arid Sheep Breeds of India.

Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane-bound G-coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out-of-season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen-2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out-of-season breeding. GG and CC genotypes were having a higher prevalence in the studied population.

Characterization of MTNR1A gene in terms of genetic variability in a panel of subtemperate and subtropical Indian sheep breeds

Seasonality of animals is an important adaptive trait for successful survival and production during limited food availability and extreme environmental conditions. Photoperiodic changes in day length are utilized by these seasonal animals as an important environmental cue for regulating their annual rhythms of reproduction cycles. Melatonin is an important hormone which is secreted by the pineal gland in proportion to darkness and its effect is mediated by melatonin receptor subtypes, principally MTNR1A. In the present study, polymorphism in the coding sequence at two important SNPs (C606T and G612A), known to be markers for out of season breeding in sheep were studied by PCR-RFLP in a panel of four breeds of sheep from subtemperate and subtropical arid conditions, respectively. The frequencies of ‘G’ and ‘A’ alleles with reference to G612A SNP did not differ considerably among all the breeds of sheep. Frequency of ‘T’ allele of the C606T SNP was found to be dominantly higher in subtemperate sheep breeds in comparison to subtropical sheep breeds. Identified SNPs in the coding region were mostly synonymous and did not lead to any change in conformation of the MTNR1A receptor protein.

Effect of short-term seclusion of sheep on their welfare indicators

Abstract Separation from the group is a potent stressor for sheep and may have an adverse effect on animal welfare. Therefore, the present study was initiated to determine the effect of short‐term seclusion of sheep on their welfare indicators in terms of behavior and endocrine level. Sixteen ewes aged 3‐4 years were kept together for one month to form a group and get emotionally accustomed to each other. Thereafter, one individual ewe was daily separated from their group and kept in a separate shed to ward off their visual and tactile contact with other group mates. Thus, the 16 ewes of the group were separated from their group individually for 24 hours one after another for one time for each individual. It was found that the dry matter intake, water intake, and body weight were reduced significantly (P < 0.05) after isolation from the group. Respiration rate, pulse rate, and rectal temperature increased significantly (P < 0.05) after separation from the group. Cortisol, thyroxine, and triiodothyronine levels were elevated after separation from the pen mate. The total time spent feeding, ruminating, and lying decreased significantly (P < 005), whereas standing and location time increased significantly (P < 0.05) after separation from the group. The percentage of time spent on different behavior at different hours also differ significantly (P < 0.05) after separation from the group as compared to when the ewes were in the group. Therefore, it can be concluded that seclusion from the group elicits a significant stress on animals, which may be due to emotional distress.

Study of circadian rhythmicity of physiological response and skin temperature of sheep during summer and winter in semi-arid tropical environment

The present study aimed to evaluate the circadian rhythmicity of some physiological variables, including rectal temperature (RT), respiration rate (RR), heart rate (HR) and skin temperature (ST) in sheep during the summer and winter in semi-arid tropical environment. Eight Malpura rams of 3-4year of age with an average body weight of 37.1±1.99kg were selected for the study. All the animals were fed individually for a maintenance requirement on a body weight basis. The RT, RR, HR and ST values were measured in each animal at 3hour intervals for 24 consecutive hours. Ambient temperature (DBT) and relative humidity (RH) was also recorded inside the shed at the time of physiological parameter study. The cosinor analysis showed a daily circadian rhythm (P<0.01) in RR, HR and ST values during the summer and winter in sheep under semi-arid environment. The RR, HR and ST differ significantly (P<0.05) between summer and winter. The amplitude of RR, RT and ST differ significantly (P<0.05) between the seasons. The results indicate that the circadian rhythmicity of RR, HR, RT and ST during summer and winter changed as expected in compliance to the seasonal requirements of animals adaptability.

Effect of thermal exposure on physiological adaptability and seminal attributes of rams under semi-arid environment

Thermal stress in hot semi-arid environment is a major limitation of sheep production in tropical and sub-tropical climatic condition. The animals tend to maintain homeostasis through physiological adjustments in a hot environment (maximum temperature reaches up to 47.5°C). Therefore, the present study was carried out to assess the effect of thermal exposure on physiological adaptability and seminal attributes of rams under semi-arid environment. The experiment was conducted for eight weeks involving sixteen Malpura crossbred rams (GMM: Garole X Malpura X Malpura). The rams were divided equally into two groups, designated as G1 and G2, respectively. The rams in G1 (Control) group were kept in a sheep shed under naturally prevailing environment without artificial manipulation of ambient temperature (Temperature 30.48±0.38°C; Relative Humidity 28.59±1.15%). The rams of G2 group were exposed to different temperature at different hours of the day (38°C at 1000-1100h; 40°C at 1100-1200h; 42°C at 12:00-1300h; 43°C at 1300-1400h; 44°C at 1400-1500h and 42°C at 1500-1600h) in a climatic chamber for thermal exposure. Physiological responses, blood biochemical profile, blood endocrine profile, sexual behavior and seminal attributes were measured for both the groups. Thermal exposure significantly (P<0.05) increased the water intake; respiration rate, rectal temperature and skin temperature at afternoon in rams. Exposure of rams to thermal stress (G2) significantly (P<0.05) increased cortisol level and decreased tri-ido-thyronine level. The latency period after the first ejaculation, decreased significantly (P<0.05) in G2. The percentage of rapid motile sperm, linearity and average path velocity of sperm were also altered significantly (P<0.05) in thermal exposed rams as compared to control. However, comparable feed intake, body weight, and major blood biochemical parameters, as well as acceptable semen quality attributes of all the rams indicated that the Fec B gene introgressed Malpura cross rams adapted to the thermal exposure under semi-arid tropical climate.