Liver infections due to amphistomes in buffaloes cause significant economic losses in livestock sector. The present study investigated the classical morphological features of adult amphistomes combined with histopathology and molecular identity in slaughtered buffaloes from Khyber Pakhtunkhwa, Pakistan. Adult amphistome were collected and morphologically characterized as Gigantocotyle explanatum. Morphometric measurement (n=50) were obtained with 12.05+-1.68 mm in length and 5.77+-0.81 mm in width, and the values of sagittal section was 6.35+-1.03 * 2.98+-0.50 mm in size. Severe bile duct pathology was observed with fibrosis of the duct wall with an irregular epithelial border, hyperplasia and inflammatory response with numerous neutrophils and eosinophils. The molecular identity of G. explanatum within Paramphistomidae was confirmed by ITS-2 rDNA sequences phylogenetic analysis based on maximum likelihood method. The genetic data based on ITS-2 secondary structure of G. explanatum consisted of four helix, Helix I, II and IV were conserved as compared with other closely related reference taxa of family Paramphistomidae and Gastrothylacidae. Helix III expressed some variations. The study concluded that rDNA ITS-2 and secondary structure information provides a guide for other researchers to determine the molecular taxonomic position of Paramphistomidae trematodes, data will support future clinical studies and control measures to reduce the amphistomiasis in buffaloes.

This trial was conducted to check the effect of commercially available hydrolysable tannin (Silvafeed Bypro) on feed intake, protein digestibility, rumen pH, ammonia nitrogen and blood urea nitrogen in Nili Ravi buffalo bulls. Four cannulated buffalo bulls were alloted in 4*4 Latin Square Design. The diet consisted of 50% seasonal fodder and 50 percent concentrate. Four iso-caloric (ME: 2800 kcal/kg) and iso-nitrogenous (CP: 18 percent) concentrate rations T1, T2, T3 and T4 were formulated and supplemented with 0, 10, 20 and 30 g hydrolysable tannin respectively per animal on daily basis. Diets as a total mixed ration were given to the bulls ad libitum twice a day. The results showed that adding tannin in diet had unaltered effect (P GT 0.05) on feed intake, digestibility of crude protein, ruminal pH, ammonia nitrogen and blood urea nitrogen. However, slightly increased numerical values were found for feed intake, crude protein digestibility and ruminal pH while slightly decreased numerical values were found for ruminal ammonia nitrogen and blood urea nitrogen at different inclusion levels of hydrolysable tannin in diet. It is concluded that numerical values for feed intake, crude protein digestibility, ruminal pH, ammonia nitrogen and blood urea nitrogen are slightly better but non-significant than control when buffalo bull were fed diets supplemented with hydrolysable tannin.

The conventional drugs used for the treatment of buffalo mastitis are losing their efficacy day by day due to increasing resistance in microbial organisms. It is therefore people nowadays are going back to use old but still quite potential remedy methods by using different herbs and shrubs for the treatment of different animal diseases including mastitis. In current investigation, three botanical extracts viz., garlic (Allium sativum L.), ginger (Zingiber officinale) and red chilies (Capsicum annuum L.) were evaluated individually and concomitantly (with ratio of 1:1) against the multidrug resistant Staphylococcus aureus and Streptococcus pyogenes isolated from buffalo mastitis. Agar well diffusion assay exhibited that red chili shown significantly (P LT 0.05) higher effects than garlic followed by ginger. All concentrations of red chili and 100 percent concentration of garlic exhibited a significantly (P LT 0.05) higher inhibitory effect against Strep. pyogenes comparing with other extracts and reference antibiotic oxacillin and streptomycin. Red chili as well as garlic in 75 and 100 percent concentrations also significantly (P LT 0.05) inhibited the Staph. aureus isolates comparing with ginger and reference drug. Red chili exhibited the highest inhibitory effects when combined with garlic than ginger. Garlic + red chilies showed a significantly (P LT 0.05) higher inhibitory effect against Staph. aureus and Strep. pyogenes as compared to other combined treatments and reference drug. Red chili as well as garlic also showed significantly (P LT 0.05) lower MIC (0.394 and 0.399 mg/ml respectively) against multidrug resistant Strep. pyogenes as compared to the ginger (0.564 mg/ml) and reference antibiotic oxacillin (0.460 mg/ml). Red chilies also showed significantly (P LT 0.05) lower (0.211 mg/ml) MIC against multidrug resistant Staph. aureus as compared to the garlic (0.391 mg/ml), ginger (0.394 mg/ml) and reference antibiotic tetracycline (0.370 mg/ml). Treatment combination based on red chili, garlic and ginger also exhibited significantly (P LT 0.05) lower MIC value against Staph. aureus and Strep. pyogenes as compared to ginger + garlic and reference antibiotic. This study concludes that red chili ranked 1st, garlic ranked 2nd and ginger ranked 3rd for antibacterial activity against multidrug resistant Staph. aureus and Strep. pyogenes. Treatment combination based on garlic + red chili ranked 1st, ginger + red chilies ranked 2nd and ginger + garlic ranked 3rd for antibacterial activity against multidrug resistant bacteria isolates.

The aim of experiment was to evaluate whether biostimulation by mature intact bull to anoestrus buffalo alters breeding performance associated with CIDR based oestrus synchronization protocol during low breeding season. Anoestrus buffalo that had calved 170.0+-10.6 (132 to 208) days before the start of the trial, were either exposed to bull (n=30, BE) or not exposed to bull (n=10, BN) for 12 days after CIDR placement (day 0). Animals received PGF 2 alpha on day 9, CIDR was removed on day 10 and Timed AI (TAI) was performed with frozen thawed semen at 48 and 60 h after CIDR removal. Buffalo were observed for oestrus detection twice daily for three days after CIDR removal. Blood sampling was done on day 9, 12 and 20 of the experiment to monitor the progesterone and cortisol levels (5 per group). Pregnancy test was done 60 days after insemination. Oestrus expression was noted in 68.97 percent (BE) and 60 percent (BN) animals (P GT 0.05). Progesterone rise above 1 ng/ml on day 20 was recorded in 4/5 (80 percent) in BE group and 2/5 (40 percent) in BN group (P GT 0.05). Conception rate was 37.9 percent (11/29) and 30 percent, (3/10) in BE and BN groups respectively (P GT 0.05). It is concluded that CIDR and fixed time artificial insemination can be used to overcome partially the problem of anoestrus and seasonality in buffalo. However, biostimulation in CIDR treated anoestrus buffalo did not increase the number of ovulating/oestrus buffalo during low breeding season probably due to a shorter exposure period (only 12 days) to bull.

This study was conducted to evaluate health care and management practices for calves less than or equal to 12 months of age in smallholder production system. Data was collected using household survey technique in Lodhran district, Punjab, Pakistan. 14 villages were selected using stratified proportionate random sampling method and 10 calf keeping households from each village were interviewed using a semi-structured questionnaire. Calves were not allowed to free range therefore all farmers practiced stall feeding. Housing facilities were of poor quality in half of farms (50.7 percent). Farmers were not using modern technologies of milk replacer, urea treated wheat straw and urea molasses blocks. All farmers offered colostrum however timings of offering differed. 75.7 percent farmers performed navel cord cutting and disinfection. All farmers offered treatment but majority (87.1 percent) practiced self-medication first. Vaccination rate was good at 94.3 percent. Almost all farmers performed drenching and dipping on their calves but there were vast differences in when and on what conditions they will be performed. Calf mortality rate for the last 12 months was 18.78 percent. Weaning age was high from modern calf rearing perspective. These findings suggest that there have been marked improvements in some parameters but farmers are still following traditional methods and practices of calf rearing due to severe lack of training related to calf rearing. There is a need for improvement in various aspects related to calf rearing including feed, housing, weaning and training.

Buffalo milk is the most preferred commodity in Pakistan due to high fat contents. Hydrolysis of circulating triglycerides and uptake of fatty acids in the mammary gland is regulated by lipoprotein lipase (LPL). This study is designed to identify single nucleotide polymorphisms (SNPs) in LPL gene among high and low milk producing buffalo breeds of Pakistan. We selected samples (n=50) of each Nili-Ravi a high milk producing and Azakheli a low milk producing buffalo breeds. Blood samples were collected for DNA extraction. LPL region of exon 2 region along with exon/intron boundaries were sequenced and data was analyzed for variation detection. Allele frequency was calculated using Hardy-Weinberg equation and in-silico analysis was performed for functional prediction and genetic diversity assessment. We found one single nucleotide polymorphism c.258+43C GT T in the intronic region of LPL gene. This polymorphism followed the Hardy-Weinberg equilibrium in both Nili-Ravi (P-value = 0.538) and Azakheli (P-value = 0.077). Association of T-allele analysis predicted it significantly higher (P-value = 0.009) in Nili-Ravi. We found one novel SNP (c.258+43C GT T) in the LPL gene in both high and low milk producing buffalo breeds of Pakistan but high in Nili-Ravi. Lower splice site effect suggests its less strength of association with milk producing trait.

To investigate the prevalence of amphistome parasites in buffaloes slaughtered at different abattoirs of Rawalpindi and Peshawar district, Pakistan. The gastro-intestinal tracts and bile ducts were examined during the period of May to December, 2017. Out of 198 buffaloes, 39 (12.8 percent) were infected with multiple species of amphistomes. In present investigation, three species of amphistomes viz Paramphistomum epiclitum, Gastrothylax crumenifer and Gigantocotyle explanatum were identified. The higher infection rate was found in Kundhi breed 13.6 percent than Azi Khali and Nili Ravi. The males buffaloes (15.1 percent) and age group of 2 to 4 years (21.4 percent) had higher infection rate as compared to females and old animals The mixed infection of amphistomes did not show significant (P GT 0.05) association among breed, sex, age, contact with water bodies and grazing management of animals. The present results will contribute to our understanding of the epidemiology of amphistomiasis in Pakistan for control and prevention studies.

Fascioliasis causes huge economic losses in livestock industry by reducing the growth, fertility rate, meat and milk yield. In the present study, somatic antigens isolated from Fasciola gigantica were assessed for the early detection of infection by developing antibody detection enzyme immunoassay. The somatic antigens extracted from adult F. gigantica worms was separated on SDS-PAGE and immunogenicity was determined by Western blot (WB). The results showed polypeptides bands between 15 to 95 kDa, and most prominent bands were 15, 28, 36, 38, 55, 72 and 95 kDa polypeptides. The WB revealed a single polypeptide of size between 36 to 55 kDa as antigenic. The sensitivity and specificity of ELISA test established for 36 to 55 kDa somatic antigens was 95.45 percent (95 percent CI: 77.16 percent to 99.88 percent) and 87.1 percent (95 percent CI: 70.17 percent to 96.37 percent), respectively. Kappa value revealed that the strength of agreement is considered to be very 'good'. In house established ELISA was implemented in the field and 12.1 percent (31/256) large ruminants were found positive, while 4.68 percent (12/256) with postmortem examination. The fasciolosis did not show significant (P GT 0.05) association with host type, breed and age groups, while significant (P LT 0.01) association with host sexes. The result confirmed that in-house established ELISA test had good value for serodiagnosis of fasciolosis in cattle and buffaloes for large scale epidemiological studies.

A retrospective analysis was conducted to examine the effect of season and climatic factors on age at puberty, service period and successful mating of Nili-Ravi buffalo maintained at a Public Sector Dairy Farm in Okara, Pakistan. Records, spread over a period of 13 years (2000 to 2012), for 583 buffaloes were analyzed for this purpose. Age at puberty was calculated by subtracting date of birth from date of first fruitful service. Average age at puberty and service period were 1056.87+-83.11 and 193.81+-47.32 days respectively. Season of birth had a significant effect (P LT 0.05) on age at puberty. Calves born in winter had a shorter age at puberty (962.09+-9.81) compared with those born in other three seasons (spring, summer and autumn). Effect of calving season on service period was significant (P LT 0.05). Autumn calvers had the shortest service period (193.81+-47.32). Service period did not differ with calving year (P GT 0.05). Month of the year and successful mating (r = 0.67) were positively correlated with each other (P LT 0.05). Maximum and minimum number of successful mating were recorded in October (21.75 percent) and June (3.88 percent). The correlation between individual climatic factors (i.e. daily temperature, rain fall, sunshine hours, relative humidity) and successful mating was non-significant (P GT 0.05). It was concluded that climatic factors did affect the reproductive activities but their individual effect was non- significant in buffalo. However, season, a combination of climatic factors, had significant effect on reproductive activities.

Paratuberculosis (PTB) in buffaloes is a chronic enteric disease triggering health implications and huge economic losses in livestock. This study was designed to explore a simple, cost-effective diagnostic approach for PTB in water buffaloes. Blood (5 ml/animal) and intestinal tissue samples accompanied by lymph nodes associated with mesentery were collected from weak and diarrhoeac animals slaughtered at local abattoirs. Out of total n=771 clinically suspected animals, only n=53 carcasses were sampled based on gross observation. Tissue smears of the gut mucosae were obtained and were made adopting special staining protocol. Tissue samples were processed by paraffin sectioning and stained with Ziehl-Neelsen and Hematoxylin-Eosin staining methods. Acid-fast bacilli were observed only in 11/53 cases on mucosal tissue smears. Pressure atrophy of small intestine villi were evident, and the mucosae were found sloughed off. The submucosae were heavily infiltrated with mononuclear cells and multifocal cellular nodules dominated by epithelioid macrophages. The foamy cytoplasm of the macrophages appeared to be engorged with acid fast bacilli and depicted the positive cases. All tissue sections of the suspected samples showed 100 percent +ve results while only 20.8 percent samples were found +ve with smear method. All histo-pathologically positive cases were further confirmed by ELISA based serological analysis. Therefore, it was concluded that histopathology is an economical and yet the most trusted tool for diagnosing bubalian PTB in countries like Pakistan.