Ecology and genetic identification of freshwater turtles in Pakistan

The turtle population plays an important role in sustaining the water ecosystem by minimizing pollution from water. The identification and molecular investigation of freshwater fauna is essential for conservation of the species that are near to extinction. The quality of water, type of flora, fauna, and environmental condition are the major factors that directly affect the distribution of freshwater turtles. Two families including eight species of freshwater turtles are found in Pakistan. The Geoemydidae (Geoclemys hamiltonii, Hardella thurjii, Pangshura smithii, and Pangshura tecta) and Trionychidae (Chitra indica, Nilssonia gangetica, Nilssonia hurum, and Lissemys punctata andersoni). Studies on the species diversity and habitat of freshwater turtle have not been focused previously in the region. The present study was the first conducted to estimate the habitat and genetic diversity of freshwater turtles using 12S rRNA (ribosomal RNA) gene in Pakistan. A total of 26 samples were collected from various localities using hand net, cast net, gills net, steel hooks, thick chemical wire, using chicken intestine and small fishes. The collected turtle specimens were morpho-taxonomically categorized into two genera, Lissemys punctata andersoni (n=13, 50%) and Nilssonia gangetica (n=13, 50%). The collected species showed an aggressive and active behavior in captivity during summer. Genomic DNA was extracted from collected specimens and used in PCR reaction by using specific primers for the amplification of short fragments of 12S rRNA gene. Analysis of generated sequences confirmed the existence of L. p. andersoni in the region. The generated sequences of L. p. andersoni correspond to Clad A and showed a close resemblance among different species of the genus Lissemys. The climatic change such as temperature and rainfall have great effects on the occurrence of turtles. Habitat degradation occurred due to various factors such as draining wetlands, deforestation, converting clear water rivers to stagnant multi-purpose reservoirs and mortality on roads when turtles move around to feed. Current study concluded that the freshwater turtles L. p. andersoni and N. gangetica are interested in natural feeds. The analysis of 359 bp of 12S rRNA gene of the genus Lissemys turtles showed relationships of these turtles with cyclanorbines flap shell turtles, which agrees with previous reports. The African taxa are paraphyletic with respect to the Asian Lissemys. The ancestors of the extant genus cyclanorbines spread from North America to Asia [26]. It should be expected that each of the 3 taxa, L. p. andersoni, L. p. punctata and L. scutata represents a distinct genetic lineage. Present molecular investigation concluded that Clad A comprising L. p. punctata, L. scutata, L. cylonensis also include L. p. andersoni species. Clad B also contains one sequence from India, identified as L. p. andersoni. Their classification as conspecific evolutionary lineages are suggested by similar genetic divergences, the observation of mismatches between morphology (spotted vs. unspotted) and mitochondrial haplotypes in clades A and B. The clades A and B provides evidence for gene flow between the spotted subspecies L. p. andersoni and adjacent populations with unspotted flap shell turtles. This study is the first investigation about the habitat and of the endemic turtle species L. p. andersoni and N. gangetica in Pakistan. The genetic identification followed by phylogenetic analysis based on 12S rRNA partial genes revealed a closest similarity with the sequences generated for the same species from the neighboring countries. This study provided information to conduct further molecular studies that are essential to provide significant genetic data about turtle species.(AU)

Molecular characterization of Paramphistomum cervi in buffaloes

Background: Paramphistomiasis (Rumen fluke disease) in ruminants is a major health problem, characterized by coarse hair, weakness, loss of appetite, weight retardations, intestine ulcers, inter-mandibular inflammation, causing substantial economic losses, and high mortality. In tropical and subtropical regions, the disease was neglected but has recently emerged as an important cause of production losses. While documented reports on Paramphistomum cervi, Paramphistomum ichikawai and Paramphistomum are limited in Asian countries and paramphistomosis has been considered the major health and economic problem in several countries. The present study aimed to identify paramphistomoid flukes that infects buffaloes with the goal of characterization of prevalence in Pakistan and its comparison with neighbor countries. Materials, Methods & Results: In 2018, a total of 178 slaughtered buffaloes aged four to six years were examined. After an immediate postmortem examination of each buffalo, flukes were collected from their infected rumen and reticulum using sterilized forceps and placed in a saline solution. DNA was extracted from adult Paramphistome species using the standard phenol chloroform method and used for amplification of partial fragment of 18S rRNA sequences using specific pair of primer. After amplification and sequencing of 18S rRNA partial fragment, the generated sequences were assembled and trimmed to remove any primer contaminations. Twenty-three randomly selected and morphologically identified adult Paramphistomum were used in species-level identification using specific primers for partial fragment of 18S rRNA sequences. The cleaned sequences (810 bp) were used to identify similar sequences using BLAST on the NCBI website. The GenBank retrieved sequences and new Paramphistomum species isolated sequences were aligned using CLUSTAL in the BioEdit Sequence...

Enhancement of extender excellence of frozen bull semen using alfa-tocopherol as an antioxidant

Background: Addition of the antioxidant to extender media is the most appropriate attempt to reduce structural lossesencounter during the process of cryopreservation. Hence semen excellence could be maintained for longer duration withoutadverse impact. Additionally antioxidants are not only capturing the reactive oxygen species but also improve the spermquality indicators and fertility. Accordingly, current elucidation has been executed to explore the dose depended appraisalof varied concentration of α-tocopherol in Tris-based extender on frozen-thawed bull semen quality parameters for enhancement of bull semen cryopreservation in the subtropical ecosystem of Peshawar.Materials, Methods & Results: Experiments were carried out on semen that has been collected from both Achai-an indigenous breed and Holstein Friesian (HF) - the exotic breed in artificial vagina maintained at 42°C from the experimentalbulls of either breed and processed independently breed wise. Semen specimens with above 70% motility were evaluatedseparately breed-wise under same environmental condition. Standard procedure was adopted to extend the collected semenin the experimental extenders and frozen subsequently. After thawing, further Analysis of the frozen straws of semen wascarried out for sperm excellence indicators that include motility, viability, acrosomal integrity and functional integrity ofspermatozoa under the subtropical condition. Sperm viability and acrosomal integrity were determined by dual stainingprocedure i.e. Trypan-blue and Giemsa stains. The hypo-osmotic swelling (HOS) test was used to assess plasma membraneintegrity. The current elucidation demonstrated that α-tocopherol 1.5 mg/mL supplemented in extender had significantly(P < 0.05) increased sperm excellence gauge that includes motilityy, viability, acrosomal integrity and functional membraneintegrity in both the breeds. On the other hand, the result further elucidated...(AU)

Enhancement of extender excellence of frozen bull semen using alfa-tocopherol as an antioxidant

Background: Addition of the antioxidant to extender media is the most appropriate attempt to reduce structural lossesencounter during the process of cryopreservation. Hence semen excellence could be maintained for longer duration withoutadverse impact. Additionally antioxidants are not only capturing the reactive oxygen species but also improve the spermquality indicators and fertility. Accordingly, current elucidation has been executed to explore the dose depended appraisalof varied concentration of α-tocopherol in Tris-based extender on frozen-thawed bull semen quality parameters for enhancement of bull semen cryopreservation in the subtropical ecosystem of Peshawar.Materials, Methods & Results: Experiments were carried out on semen that has been collected from both Achai-an indigenous breed and Holstein Friesian (HF) - the exotic breed in artificial vagina maintained at 42°C from the experimentalbulls of either breed and processed independently breed wise. Semen specimens with above 70% motility were evaluatedseparately breed-wise under same environmental condition. Standard procedure was adopted to extend the collected semenin the experimental extenders and frozen subsequently. After thawing, further Analysis of the frozen straws of semen wascarried out for sperm excellence indicators that include motility, viability, acrosomal integrity and functional integrity ofspermatozoa under the subtropical condition. Sperm viability and acrosomal integrity were determined by dual stainingprocedure i.e. Trypan-blue and Giemsa stains. The hypo-osmotic swelling (HOS) test was used to assess plasma membraneintegrity. The current elucidation demonstrated that α-tocopherol 1.5 mg/mL supplemented in extender had significantly(P < 0.05) increased sperm excellence gauge that includes motilityy, viability, acrosomal integrity and functional membraneintegrity in both the breeds. On the other hand, the result further elucidated...

Diet composition and seasonal fluctuations in the feeding habit of snow barbel (Schizothorax plagiostomus) in River Indus, Pakistan

Background: Schizothorax plagiostomus is widely distributed in river Indus and is most important food fish in Pakistan. The feeding habit of fish is directly related to the size of fish, its metabolic rate and environmental temperature. The accurate description of fish diet and feeding habit is a very important aspect in fisheries management for the purpose of species conservation, breeding and culture. The present work was aimed to investigate the specie abundance, the diet composition and seasonal variations in the feeding habit of Snow barbell Schizothorax plagiostomus.Materials, Methods & Results: A total of 1799 fish specimens were caught at the confluence of six tributaries along river Indus at Indus Kohistan, northeastern Pakistan. The fish were collected by 5-panels of gill net during first week of each month. The site specific Catch Per Unit Effort (CPUE) and season specific CPUE of fish fauna were assessed. For the gut content analysis 240 samples (99 male and 141 females) of S. plagiostomus were selected on monthly basis. Frequency of occurrence method and volumetric method were applied to record the different food items in the gut of S. plagiostomus. The physico-chemical parameters, NO3 concentration and dissolved Co2 of water from different localities of river Indus were recorded month wise by Hach sensION 156 meter, Horiba LAQUA Nitrate Meter and EA80 meter respectively. Significant difference was observed in water temperature during the four seasons. Except alkalinity no other water parameter showed significant variation across different localities. The results showed that highest Mean CPUE was observed for Darel Stream (0.55) and lowest for Jalkot stream (0.26). Peak abundance of fish was recorded in the month of November with a mean catch of 44.50, mean CPUE of 0.74 and mean Kruskal-Wallis rank value of 63.25.[...]

Diet composition and seasonal fluctuations in the feeding habit of snow barbel (Schizothorax plagiostomus) in River Indus, Pakistan

Background: Schizothorax plagiostomus is widely distributed in river Indus and is most important food fish in Pakistan. The feeding habit of fish is directly related to the size of fish, its metabolic rate and environmental temperature. The accurate description of fish diet and feeding habit is a very important aspect in fisheries management for the purpose of species conservation, breeding and culture. The present work was aimed to investigate the specie abundance, the diet composition and seasonal variations in the feeding habit of Snow barbell Schizothorax plagiostomus.Materials, Methods & Results: A total of 1799 fish specimens were caught at the confluence of six tributaries along river Indus at Indus Kohistan, northeastern Pakistan. The fish were collected by 5-panels of gill net during first week of each month. The site specific Catch Per Unit Effort (CPUE) and season specific CPUE of fish fauna were assessed. For the gut content analysis 240 samples (99 male and 141 females) of S. plagiostomus were selected on monthly basis. Frequency of occurrence method and volumetric method were applied to record the different food items in the gut of S. plagiostomus. The physico-chemical parameters, NO3 concentration and dissolved Co2 of water from different localities of river Indus were recorded month wise by Hach sensION 156 meter, Horiba LAQUA Nitrate Meter and EA80 meter respectively. Significant difference was observed in water temperature during the four seasons. Except alkalinity no other water parameter showed significant variation across different localities. The results showed that highest Mean CPUE was observed for Darel Stream (0.55) and lowest for Jalkot stream (0.26). Peak abundance of fish was recorded in the month of November with a mean catch of 44.50, mean CPUE of 0.74 and mean Kruskal-Wallis rank value of 63.25.[...](AU)

A revision of two distinct species of Rhipicephalus: R. microplus and R. australis / Uma revisão sobre duas espécies distintas de Rhipicephalus: R. microplus e R. Australis

Rhipicephalus (Boophilus) species are monoxenous ticks with seasonal distribution in tropical and subtropical regions. For many years, Rhipicephalus microplus was considered as a single species; however, further analysis split these ticks into two distinct species. Because R. microplus and R. australis share similar attributes, it is hard to discriminate these two species and explain the changes in the classification of these parasites over the past decades. The reappearance of R. australis is an outcome of new research, which has afforded to better characterize these probably cryptic species. Evidence based on morphological features, the lack of conspecificity, microsatellite markers, mitochondrial 12S and 16S ribosomal DNA, and mitochondrial genome supports the re-classification of R. microplus as different species. Therefore, populations of R. microplus from Australia, Cambodia, Philippines, Indonesia, New Caledonia, Borneo, New Guinea, Tahiti and parts of Southeast Asia were recently reinstated as R. australis . Moreover, a better knowledge on the speciation between these two species could pave the way to important advances in tick control strategies.(AU)

As espécies pertencentes ao gênero Rhipicephalus (Boophilus) são carrapatos monoxenos de distribuição sazonal em regiões tropicais e subtropicais. Por muito anos, duas espécies de carrapatos foram consideradas como Rhipicephalus microplus. Contudo, estudos recentes reclassificaram esse carrapato em duas espécies: R. microplus e R. australis . Em razão de diversas semelhanças entre R. microplus e R. australis, distinguir essas duas espécies torna-se uma tarefa árdua, o que explica as mudanças de classificação dessas espécies nas últimas décadas. O reaparecimento da espécie R. australis surge com novas pesquisas, resultado de uma melhor caracterização dessas prováveis espécies crípticas. Evidências baseadas em análises das características morfológicas, na ausência de co-especificidade, em marcadores de microssatélites, no DNA ribossomal mitocondrial 12S e 16S, assim como no genoma mitocondrial, suportam a re-classificação de R. microplus como duas espécies distintas. Nesse sentido, populações de R. microplus da Austrália, Camboja, Nova Caledônia, Bornéo, Filipinas, Nova Guiné, Indonésia e Taiti foram recentemente renomeadas como R. australis . Além disso, um melhor entendimento sobre a especiação e localização dessas duas espécies pode trazer avanços importantes para melhorar as estratégias de controle desses carrapatos.(AU)

Molecular characterization of Echinococcus species in Khyber Pakhtunkhwa, Pakistan

Background: Species belonging to genus Echinococcus are cestode parasites well known for helminthic infections globally.Diseases caused by these parasites are serious health threats for public and veterinary sectors. DNA-based characterization confirmed genetic variability among Echinococcus species and resulted in the identification of 10 genotypes (G1-10).Among identified Echinococcus species, E. granulosus and E. multilocularis are clinically most important responsiblefor cystic echinococcosis and alveolar echinococcosis, respectively. Identification and genetic characterization of thesecestode parasites at species level is essential for disease diagnosis and control measures. This study aimed at narrowinggap of missing knowledge on Echinococcus spp. and their genotypes in Khyber Pakhtunkhwa (KP), Pakistan.Materials, Methods & Results: Hydatid cysts of human source were obtained under aseptic conditions from thoracic surgery unit of the Lady Reading Hospital (LRH) at Peshawar, KP, Pakistan. Hydatid cysts from animal source (cattle) werecollected at Peshawar visiting numerous abattoirs. Theses cyst samples (n = 40) were collected from animals (cattle) (n= 30) and human sources (n = 10). Nucleic acid was extracted from aspirates obtained from cysts, and investigated usingpolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of a mitochondrial coding gene(rrnL, large subunit of ribosomal RNA). A pair of primers (ECH-LSU/F and ECH-LSU/R) were used to amplify a 570-bpDNA fragment of a mitochondrial gene (rrnL, large subunit of ribosomal RNA) containing a species-specific SspI restriction site for the differentiation among E. granulosus and E. multilocularis. Overall results indicated that among the cystscollected from animal and human sources, majority were positive for E. granulosus (n = 24, 60%) and E. multilocularis (n= 16, 40%)...(AU)

Molecular characterization of Echinococcus species in Khyber Pakhtunkhwa, Pakistan

Background: Species belonging to genus Echinococcus are cestode parasites well known for helminthic infections globally.Diseases caused by these parasites are serious health threats for public and veterinary sectors. DNA-based characterization confirmed genetic variability among Echinococcus species and resulted in the identification of 10 genotypes (G1-10).Among identified Echinococcus species, E. granulosus and E. multilocularis are clinically most important responsiblefor cystic echinococcosis and alveolar echinococcosis, respectively. Identification and genetic characterization of thesecestode parasites at species level is essential for disease diagnosis and control measures. This study aimed at narrowinggap of missing knowledge on Echinococcus spp. and their genotypes in Khyber Pakhtunkhwa (KP), Pakistan.Materials, Methods & Results: Hydatid cysts of human source were obtained under aseptic conditions from thoracic surgery unit of the Lady Reading Hospital (LRH) at Peshawar, KP, Pakistan. Hydatid cysts from animal source (cattle) werecollected at Peshawar visiting numerous abattoirs. Theses cyst samples (n = 40) were collected from animals (cattle) (n= 30) and human sources (n = 10). Nucleic acid was extracted from aspirates obtained from cysts, and investigated usingpolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of a mitochondrial coding gene(rrnL, large subunit of ribosomal RNA). A pair of primers (ECH-LSU/F and ECH-LSU/R) were used to amplify a 570-bpDNA fragment of a mitochondrial gene (rrnL, large subunit of ribosomal RNA) containing a species-specific SspI restriction site for the differentiation among E. granulosus and E. multilocularis. Overall results indicated that among the cystscollected from animal and human sources, majority were positive for E. granulosus (n = 24, 60%) and E. multilocularis (n= 16, 40%)...

A study on vas deference and seminal vesicle of male goats (Capra hircus)

Background: Reproductive activity of male animal is strongly associated with development of the testicle, the conductor channels and accessory sex gland. Vas deference may be considered the extra-testicular continuation from cauda of the epididymis, and it is the portion of the reproductive tract fundamentally associated with transportation of the sperm-containing fluid from each epididymis to the urethra for their finishing discharge. Non-ampulated part is further divided into: extra-abdominal portion along the caudal border of testes, up to the vaginal ring and abdominal portion started from the vaginal ring to ampullae. Accessory sex glands including vesicular gland and ampullae of vas deferens are characteristically essential for reproductive process. Gross morphometric features of anatomical structures of male reproductive tract in an animal make available a very valuable mechanism in understanding of several physiological and reproductive phenomena. The current study aim at documenting baseline data on gross morphometric aspects of vas deference and seminal vesicle in adult male local nondescript goats in Pakistan ecology that could be utilized as reference values in evaluating their congenital defects and gross pathological abnormalities.Materials, Methods & Results: Experiments were carried out on ampullated and non-ampullated segment of vas deference and seminal vesicle. A total of n = 100 local male goats of 2-3 years of age were selected for this study. Vas deference and seminal vesicles (accessory sex gland) were collected immediately after slaughter from local abattoirs. The non-ampullated segment of vas deference was further divided into abdominal and extra-abdominal segment. Specimens were dissected and washed with normal saline. Standard procedure was adopted for these morphometric features of vas deference and seminal vesicle by using vernier caliper, scale, non-stretchable thread and electronic weighing balance.[...](AU)

A study on vas deference and seminal vesicle of male goats (Capra hircus)

Background: Reproductive activity of male animal is strongly associated with development of the testicle, the conductor channels and accessory sex gland. Vas deference may be considered the extra-testicular continuation from cauda of the epididymis, and it is the portion of the reproductive tract fundamentally associated with transportation of the sperm-containing fluid from each epididymis to the urethra for their finishing discharge. Non-ampulated part is further divided into: extra-abdominal portion along the caudal border of testes, up to the vaginal ring and abdominal portion started from the vaginal ring to ampullae. Accessory sex glands including vesicular gland and ampullae of vas deferens are characteristically essential for reproductive process. Gross morphometric features of anatomical structures of male reproductive tract in an animal make available a very valuable mechanism in understanding of several physiological and reproductive phenomena. The current study aim at documenting baseline data on gross morphometric aspects of vas deference and seminal vesicle in adult male local nondescript goats in Pakistan ecology that could be utilized as reference values in evaluating their congenital defects and gross pathological abnormalities.Materials, Methods & Results: Experiments were carried out on ampullated and non-ampullated segment of vas deference and seminal vesicle. A total of n = 100 local male goats of 2-3 years of age were selected for this study. Vas deference and seminal vesicles (accessory sex gland) were collected immediately after slaughter from local abattoirs. The non-ampullated segment of vas deference was further divided into abdominal and extra-abdominal segment. Specimens were dissected and washed with normal saline. Standard procedure was adopted for these morphometric features of vas deference and seminal vesicle by using vernier caliper, scale, non-stretchable thread and electronic weighing balance.[...]