A novel selection signature in stearoyl-coenzyme A desaturase (SCD) gene for enhanced milk fat content in Bubalus bubalis.

Modern molecular interventions are dynamic gears for breeding animals with superior genetic make-up. These scientific efforts lead us toward sustainable dairy herds with improved milk production in terms of yield and quality. Many of candidate genes have been dissected at molecular level, and suitable genetic markers have been identified in cattle, but this work has not been validated in buffaloes so far. Stearoyl-coenzyme A desaturase (SCD) has been a potential candidate gene for fat content of milk. Genomic analysis of SCD revealed a total of six variations that were identified through DNA sequencing of animals with lower and higher butter fat %age. After statistical analysis, genotype AB of p.K158I could be associated (P value <0.0001) with higher milk fat %age (10.5 ± 0.5464). This SNP was validated on larger data set by cleaved amplified polymorphic sequences (CAPS) by using DdeI. To scrutinize the functional consequences of p.K158I, 3D protein structure of SCD was predicted by homology modeling and this variation was found located in the vicinity of functional domain and a part of transmembrane helix of this membrane integrated protein. This is a first report toward genetic screening of SCD gene at molecular level in buffalo. This report illustrates the implication of SCD gene and in particular p.K158I variation, in imparting its effect on milk fat %age, which can be targeted in selection of superior dairy buffaloes.

Mutational spectrum of the TYR and SLC45A2 genes in Pakistani families with oculocutaneous albinism, and potential founder effect of missense substitution (p.Arg77Gln) of tyrosinase.

BACKGROUND: Oculocutaneous albinism (OCA) is an autosomal recessive disorder of abnormal melanin formation, which results in hypopigmentation of skin, hair and eyes. OCA is classified into four types based on clinical and genetic findings. OCA1 is the most severe form of albinism, and is caused by mutations in the tyrosinase (TYR) gene, while OCA4 is caused due to mutations in SLC45A2. METHODS: In total, 13 families with ≥ 3 members with OCA were enrolled. Family history was ascertained and pedigrees were drawn up. Blood samples were collected and processed for DNA extraction. Linkage analysis was performed by typing three short tandem repeat markers in candidate regions of TYR and SLC45A2. Sequence analysis was performed of all the coding exons and adjacent intronic sequences of both genes. RESULTS: Eight families showed linkage to OCA1 and one family showed linkage to OCA4. Four missense substitutions (p.Arg239Trp, p.Ser192Tyr, p.Ser44Arg and p.Arg77Gln) were identified in TYR in the families with OCA1 linkage, and another missense substitution (p.Gln272Lys) was identified in the family with OCA4 linkage. One of the identified missense substitution (p.Arg77Gln) in TYR was found in five different families, which had a common haplotype. CONCLUSIONS: We identified four missense substitutions in TYR and a single missense substitution in SLC45A2. One missense substitution (p.Arg77Gln) in TYR was found in five different families that originated from the same geographical area and displayed a common haplotype, suggesting a single origin that then spread to different geographical areas of Azad Kashmir, Pakistan.

Identification of a novel mutation (p.Ile198Thr) in gene TYR in a Pakistani family with nonsyndromic oculocutaneous albinism.

The TYR gene (MIM #6069333) is located at position 11q14.3 on the human chromosome, and encodes tyrosinase, which is expressed in melanocytes and controls the biosynthesis of melanin. Most TYR mutations eliminate the activity of tyrosinase, preventing melanocytes from producing any melanin throughout life. People with this form of albinism have white hair, light-coloured eyes and very pale skin. Some mutations in TYR reduce but do not completely eliminate tyrosinase activity, and allow some melanin to be produced. We report a Pakistani family with four members affected by oculocutaneous albinism (OCA). Blood samples were collected from all affected individuals, normal siblings and their parents. Genomic DNA was extracted, and sequence analysis of all the coding exons and adjacent intronic sequences of TYR was performed, which identified a novel missense substitution (p.Ile198Thr). Sequencing of TYR in 90 unrelated healthy individuals showed no sequence variant at this location. Our study expands the mutational spectrum of OCA1.

Mitochondrial DNA diversity patterns in Pakistani buffalo.

The Indian subcontinent is considered to be the likely centre of river buffalo domestication, based on population dynamics, archaeological evidence and genetic diversity. Recent studies on mitochondrial DNA diversity have drawn useful conclusions about the domestication history of Bubalus bubalis. The conclusions of these studies are, however, incomplete, unless samples can also be analysed from Pakistan, which contains the second largest buffalo population of the world. Here, we report the results of the first study on mitochondrial D-loop sequence diversity in five breeds of Pakistani buffalo. Analysis of sequence variations in 503-bp of the D-loop region of 123 animals revealed 52 haplotypes, including 40 singletons. Multidimensional display of breed pairwise F(ST) values revealed no strong clustering of breeds. Bayesian, maximum parsimony, neighbour joining and UPGMA trees revealed a topology consistent with domestication as well as subsequent introgression of multiple maternal lineages from the wild stocks. Reduced median network analysis provided evidence of population expansion from more than one set of haplotypes. The study also confirmed that Pakistani buffalo are of the river type. The observed mitochondrial D-loop sequence diversity suggests that Pakistani areas bordering India might have contributed to the initiation of domestication of the present-day river buffalo.

Genetic variants in interferon gamma (IFN-γ) gene are associated with resistance against ticks in Bos taurus and Bos indicus.

In tropical and subtropical regions of the world, parasitic diseases are major obstacle in the health and ultimately overall performance of animals. Cattle express heritable, contrasting phenotypes when exposed to ticks depicting genetic nature of trait. IFN-γ is one of the most reported genes critical for innate and adaptive immunity against viral and intracellular infections. To identify its role in resistance for ectoparasite especially tick, genetic characterization of this gene was done in resistant and susceptible animals of Sahiwal cattle (n = 95) and Friesian (n = 92). Nine Polymorphisms were identified, three of them were found in exonic region. One out of nine variants was being reported previously (ss82716193) and was confirmed in Pakistani Sahiwal cattle population as well. Single site analysis of each variant depicted their significance in tick resistant and tick susceptible groups (P < 0.05). The associations using haplotypes were more informative than for single markers. Eighteen different haplotypes resulting from nine polymorphic sites were used in construction of maximum parsimony tree which categorized resistant and susceptible animals in two clades. Genetic markers identified in this study can be useful in future breeding selection programs against tick resistance.

Gamma radiation induced mutagenesis in Aspergillus niger to enhance its microbial fermentation activity for industrial enzyme production.

α- and ß-Galactosidases find application in food processing, health and nutrition. Aspergillus niger is one of the potent producer of these enzymes and was genotypically improved using gamma-ray induced mutagenesis. The mutant-derivative produced two-fold higher α- and ß-galactosidases. For testing genetic variability and its relationship with phenotypic properties of the two organisms, DNA samples of the mutant and parental strains of A. niger were amplified with 28 deca-nucleotide synthetic primers. RAPD analysis showed significantly different pattern between parental and mutant cultures. The mutant derivative yielded homogeneous while parental strain formed heterogeneous amplification patterns. Seven primers identified 42.9% polymorphism in the amplification products, indicating that these primers determined some genetic variability between the two strains. Thus RAPD was found to be an efficient technique to determine genetic variability in the mutant and wild organisms. Both wild and mutant strains were analyzed for their potential to produce galactosidases. Comparison of different carbon sources on enzyme yield revealed that wheat bran is significant (P < 0.01) effective producer and economical source followed by rice bran, rice polishing and lactose. The mutant was significantly better enzyme producer and could be considered for its prospective application in food, nutrition and health and that RAPD can be effectively used to differentiate mutant strain from the parental strain based on the RAPD patterns.

Influence of varying dietary electrolyte balance on broiler performance under tropical summer conditions.

A 6-week feeding trial was conducted to determine the effects of varying dietary electrolyte balance (DEB) on growth performance and physiological responses in broiler chicks reared during hot summer months (26.1-37.5 degrees C). The average minimum and maximum room temperatures recorded from 14 to 42 days of age were 26.1 and 37.5 degrees C, respectively, with relative humidity ranging from 51% to 55%. Five DEB treatments supplying 0, 50, 150, 250 and 350 (Na(+)+K(+)-Cl(-)) mEq/kg of diet were prepared by adding NaHCO(3) and/or NH(4)Cl to basal diet that contained 185 mEq/kg from 1 to 28 days and 172 mEq/kg from 29 to 42 days. These DEB diets were randomly offered to six experimental units of 20 chicks each. Improved 42-day body weight gain (BWG), feed:gain (F:G) and reduced mortality was noted with DEB 50, 150 and 250 mEq/kg compared to DEB 0 and 350 mEq/kg. A quadratic effect of increasing DEB level was observed on BWG and F:G, whereas the effect was linear for feed intake, water intake and litter moisture. The DEB 50, 150 and 250 maintained better blood pH, pCO(2) and HCO(3) concentrations during acute heat stress periods than that of 0 and 350. The blood heterophil and lymphocyte percent as well as heterophil:lymphocyte were remained unaffected. The lowest DEB (0 mEq/kg) diet reduced the feed intake and promoted metabolic acidosis. At 350 mEq/kg DEB, water intake and metabolic alkalosis were promoted probably due to high levels of dietary NaHCO(3). The study concluded that overall better performance was recorded with DEB 50, 150 and 250 mEq/kg. These results indicated that single optimal DEB value could not be recommended to combat heat stress in broilers.

Frequencies of PrP genotypes and their implication for breeding against scrapie susceptibility in nine Pakistani sheep breeds.

Prion protein (PrP) gene of 308 sheep was genotyped to investigate polymorphisms at scrapie-associated codons 136, 154 and 171 to assess the resistance of nine different Pakistani sheep breeds to natural/typical scrapie. As a result six genotypes were established on the basis of polymorphic codons 154 and 171. The most scrapie-susceptible codon 136 (A/V) was monomorphic (A) in all breeds. Wild-type genotype ARQ/ARQ was detected with maximum prevalence ranging from 63.2% in crossbred Pak-karakul to 100% in native Buchi, Kachi and Thalli breeds. The most frequent of typical scrapie-associated genotypes was ARQ/ARR as indicated by five of nine breeds. The coding region of PrP gene of 49 animals from the total sampled was also sequenced to ascertain additional polymorphisms. Polymorphism was found in 13 animals of the six breeds in codons 101(Q/R), 112(M/T), 146(N/S) and 189(Q/L) and ten genotypes were established on the basis of these polymorphic codons. Only Hissardale possessed five of the ten genotypes. The most frequent genotype was M(112)ARQ/T(112)ARQ detected in Hissardale, Pak-karakul and Awassi, whereas genotypes ARQr(231)/ARQr(231) and ARQR(231)/ARQr(231) (established on the basis of silent polymorphism agg/cgg-R/R) were detected in all breeds. Some animals consisted of three polymorphisms at different PrP codons that are not common in European breeds. An infrequent double heterozygosity (c/c a/g g/t) for codon 171 resulting in a genotype R/H was also detected in three animals each one from Kajli, Hissardale and Pak-karakul. This study concludes that all native sheep breeds are poor in scrapie-resistant PrP genotypes and could contract scrapie if exposed to prions.

Fluorescent in-situ hybridization of cattle and sheep chromosomes with cloned human fragile-X DNA.

An extensive study on spontaneous and 5-Fluorodeoxyuridine induced fragile sites identified Xq31 in cattle (Bos taurus) and (Xq24, Xq26) in sheep (Ovis aries) in addition to several autosomal fragile sites (under publication). A ZOO-FISH study using three cloned human fragile-X probes with CCG/CGG(n) trinucleotide repeat sequence was carried out to determine homology between human and bovine fragile-X. The hybridisation results showed only a weak signal on a human chromosome that was not an X with all three fragile site probes. No signals were detected in sheep chromosomes. The signal of all three human fragile-X probes on cattle chromosomes was however, medium-prominent sub-centromeric signal on two homologues. BrdU administration in 12 h before harvesting identified these homologues to be chromosome number 5. In addition retrospective slides of cattle and sheep chromosomes used for fragile site studies showed no signals whatsoever. It was therefore concluded that no homology existed between human and bovine fragile-X.

Prion protein gene polymorphisms in four goat breeds of Pakistan.

Four different goat breeds (Pak-Angora, Dera Din Panah, Naachi and Teddy) of Pakistan were selected to investigate polymorphism in the prion protein gene (PrP gene) responsible for scrapie disease resistance in goats. Initially, genotyping of 187 animals of these four breeds by restriction fragment length polymorphism (RFLP) was done to see the genotype for codon 136 and 154. All the animals were monomorphic with a genotype of AARR except one animal of Teddy breed having the genotype of AARH. Sequencing of PrP gene of twenty animals representing these four goat breeds revealed two genotypes PPSSSS and PPSSPS with haplotypes PSS and PSP of PrP gene at the codon numbers 42, 138, and 240. All four breeds showed both wild type monomorphic sequence and mutant polymorphic sequences of these codons. The mutants of 42 and 138 codons translate the same amino acids as with the wild type sequences, while the mutant of codon 240 is responsible for a different amino acid translation i.e., serine to proline. In short, this study provides preliminary information about alleles and genotypes of PrP gene in four goat breeds of Pakistan.

Effect of potassium chloride supplementation in drinking water on broiler performance under heat stress conditions.

The effect of water supplementation of KCl on performance of heat-stressed Hubbard broilers was evaluated in the present experiment. The 3 experimental treatments (i.e., control, 0.3 and 0.6% KCl) were allocated to 3 replicates of 15 birds each. The control group was kept on dugout tap water, whereas the other 2 groups were supplied water supplemented with 0.3 and 0.6% KCl (wt/vol) by supplementing 3 and 6 g of KCl, respectively, per liter of drinking water. Broilers were provided ad libitum access to feed and water for the experimental period of 7 to 42 d of age and kept in open-sided house. The birds were reared under continuous thermostress (minimum 28.2 +/- 1.02 and maximum 37.5 +/- 0.78 degrees C) environment. Supplementing drinking water with 0.6% KCl reduced panting-phase blood pH to 7.31 and significantly increased live BW gain by 14.5 (P = 0.036) and 7.9% (P = 0.029) at 28 and 42 d of age, respectively, relative to control. An improved (P = 0.04) feed:gain and lowered body temperature were noted in groups supplemented with 0.6% KCl as compared with control and 0.3% KCl. Enhanced physiological adaptation with 0.6% KCl was evidenced by a more favorable pH during the panting phase in the present study. These findings demonstrated a possibility of better broiler live performance through KCl supplementation under conditions of severe heat stress (35 to 38 degrees C).

Effect of low-protein diets having constant energy-to-protein ratio on performance and carcass characteristics of broiler chickens from one to thirty-five days of age.

A trial was conducted to determine the effect of low-protein diets with constant ME:CP ratio on performance and carcass characteristics of broilers from 1 to 35 d of age. Four experimental diets were formulated to have 4 levels of CP and ME, respectively, in each phase: 23, 22, 21, and 20% CP with 3,036, 2,904, 2,772, and 2,640 kcal/kg in the starter phase (1 to 10 d); 22, 21, 20, and 19% CP with 3,146, 3,003, 2,860, and 2,717 kcal/kg in the grower phase (11 to 26 d); and 20, 19, 18, and 17% CP with 3,100, 2,945, 2,790, and 2,635 kcal/kg in the finisher phase (27 to 35 d). Digestible Lys was maintained at 1.10, 1.02, and 0.90% of the diet in the starter, grower, and finisher periods, respectively. A total of 1,760 one-day-old Hubbard broiler chickens were randomly divided into 16 experimental pens, 110 chickens in each pen, and each diet was offered to 4 replicates at random. Weight gain was linearly decreased (P < 0.001), whereas feed intake and feed conversion ratio were increased (P < 0.001) linearly as dietary protein and energy decreased during grower, finisher, and overall experimental periods. Protein efficiency ratio and energy efficiency ratio were decreased (P < 0.05) with low-CP and low-ME diets during the grower, finisher, and overall experimental period. However, carcass yield, breast meat yield, thigh yield, abdominal fat, and relative liver and heart weights were not affected by the treatments. Feeding broiler chickens low-CP diets with constant ME:CP ratio has adversely affected the growth performance, but carcass parameters were unaffected without any increase in abdominal fat content.