Nocturnal variation of prolactin secretion in the Mouflon (Ovis gmelini musimon) and domestic sheep (Ovis aries): seasonal changes

Seasonal changes in nocturnal prolactin secretion and their relationship with melatonin secretion were monitored in wild (Mouflon, Ovis gmelini musimon) and domesticated sheep (breed Manchega, Ovis aries). Two groups of eleven adult females each, were maintained outdoors under natural photoperiod. Plasma concentrations of prolactin and melatonin were determined during the summer and winter solstices and the autumn and spring equinoxes. Blood samples were collected every 3h during the night hours, and 1h before and after the onset of darkness and sunrise. Maximum mean plasma concentrations of prolactin during the dark-phase in Mouflons were observed in the summer solstice, (P<0.001) and in the summer solstice and spring equinox in Manchega ewes (P<0.001). Mean plasma concentrations of prolactin were higher in the wild species (P<0.001) during the summer solstice. In contrast, during the spring equinox, mean levels of prolactin were higher in Manchega ewes than in Mouflons (P<0.05). Plasma prolactin concentrations showed a nocturnal rhythm in both breeds, with seasonal variations (P<0.001). The increase in plasma melatonin levels during the first hour after sunset was accompanied to increasing concentrations of PRL 1h after the onset of darkness, only in the autumn and spring equinox for the Mouflon, and in the summer solstice and spring equinox for the Manchega ewes. In Mouflons, the fall of plasma PRL concentrations about the middle dark-phase in all the periods studied, coincided with high levels of melatonin. A similar relation was observed in Manchega ewes only in the winter solstice and spring equinox. The current study shows that the nocturnal rhythm of prolactin secretion exhibits seasonal variation; differences in the patterns of prolactin secretion between Mouflon and Manchega sheep are taken to represent the effects of genotype.     

Ecology and behaviour of domestic sheep (Ovis aries): a review

Behaviour of sheep is complex and poorly understood. It is only within the past 20 years that a concerted effort has been made to elucidate those aspects of behaviour which have most relevance to sheep raising under open-range conditions. The present report attempts to summarize and review published work on the behaviour and ecology of sheep. The whole question of the significance of behaviour and the extent to which it is possible to shape it is a field which is open for study. This paper is an attempt to summarize the work on sheep behaviour and on those aspects of physiology which impinge. Consideration is given to the evolution of Bovids, scope of domestication and world distribution of sheep since these aspects have a major influence on the sheep with which we deal today. Animal requirements for food, water, shelter, protection from predators, and control of disease and parasites have been largely met by man and the forces for selection which operated among the progenitors of domestic sheep have been largely negated. Despite this, genetic selection still goes on. Behavioural adaptation to a wide variety of environmental and nutritional regimes is a common feature among free-ranging sheep. Reproductive success, a matter of considerable commercial concern is often poor. Lamb survival is of critical importance. Factors involved in neonatal mortality are reviewed in detail. Finally, the now extensive literature on sheep behaviour, physiology and management is collated in a bibliography with over 150 literature citations.     

Prolactin inhibition does not influence horn growth in two wild caprinae species: European mouflon (Ovis orientalis musimon) and Iberian ibex (Capra pyrenaica)

Studies on seasonal horn development and the endocrine mechanism regulating its pattern in wild ruminants are scarce. The aim of this paper was to study the influence of photoperiod and prolactin (PRL) on horn growth in two wild ruminant species: the European mouflon and the Iberian ibex. Eighteen male ibexes and 13 mouflon rams, maintained in captivity, were divided into three groups: a control group, kept under a natural photoperiod (latitude, 40°25′ N); a long-day group, exposed to an artificial photoperiod of 15-h light and 9-h darkness; and a group treated with bromocriptine (BCR; 10?mg twice weekly during spring and summer) to induce hypoprolactinaemia. Horn length growth (HLG) was recorded weekly for 18?months; plasma PRL concentrations were measured twice monthly by radioimmunoassay. In the ibexes of the long-day group, the period of strong horn development during spring–summer was significantly reduced by 2?months compared with the controls. In the mouflons of the long-day group, this same period was significantly increased by 9?months. In the BCR-treated animals, hypoprolactinaemia was observed in both species, but HLG was the same as in the corresponding controls. The present results suggest that the seasonal pattern of horn growth of wild ruminants is primarily modulated by photoperiod in a species-dependent manner. The persistence of resurgence of horn growth during spring in the BCR-induced hypoprolactinaemic animals of both species suggests that annual variations in blood PRL concentration have no effect on seasonal variation in horn growth.     

Interspecific embryo transfer from mouflon (Ovis gmelini musimon) to domestic Corriedale sheep in Argentina

An interspecific embryo transfer program was conducted for genetic improvement and increasing the number of offspring from a flock of mouflon sheep in Argentina. The female donor mouflons were divided into three groups, G1 (n=5), G2 (n=4) and G3 (n=5). The total NIH-FSH-P1 dose given to each donor on the superovulatory treatment was 260, 200 and 160 mg for G1, G2 and G3, respectively. The mouflons in G3 were maidens, while the others were multiparous. Domestic Corriedale ewes (n=60) were synchronized and used as recipients. The embryo recovery and transfer was performed by a surgical method. Mouflons (n=13) responded to the superovulatory treatment with an average of 9.1+/-2.8 ovulations. A low incidence of early luteal regression was found (1 out of 14 donors). Embryo recovery rates were 60, 31 and 76% in groups G1, G2 and G3, respectively. The percentage of transferable embryos obtained in G1 and in G2 exceeded 80%. None of the embryos obtained from G3 were of transferable quality. In G1, 25 transferable embryos were recovered and transferred to 13 recipients, resulting in a pregnancy rate of 76.9% (10/13). In G2, 10 embryos were transferred to 5 recipients, resulting in a 60% pregnancy rate (3/5). Lambing rate was 60% (15/25) and 30% (3/10) for G1 and G2, respectively. Thirteen lambs were born to the 14 donors following natural service after the embryo recoveries. This study demonstrates that the application of IET technology would have great reproductive impact, especially when the donor mouflon hinds are selected according to age and reproductive history.     

Embryo recovery from superovulated mouflons (Ovis gmelini musimon) and viability after transfer into domestic sheep

In this study multiple ovulation and embryo transfer (MOET) technology was tested as a method for increasing the number of offspring obtained from superovulated mouflons and then using Sardinian ewes as recipients. Two experiments were carried out over consecutive years. In Experiment 1, female mouflons received a standard superovulatory treatment during both breeding and anoestrous seasons. Sarda sheep, used as controls, received the same treatment. Mean superovulatory response (corpora lutea and large follicles) was higher in the domestic sheep than in the mouflons (4.8 vs. 10.1 and 4.2 vs. 8.8 in breeding and anoestrous seasons, respectively; P < 0.05). A high percentage of mouflons showed early luteal regression which negatively affected recovery rate (35% and 30% in mouflons vs. 69% and 71% in sheep) and the yield of embryos suitable for transfer (37% and 25% in mouflons vs 74% and 69% in sheep; P < 0.05). In Experiment 2, ten mouflons were treated by the same superovulatory protocol and divided into two groups. In the first (Group 5), embryos were recovered earlier by oviductal flushing and cultured in vitro with oviductal cells in CZB medium until the morula/blastocyst stage; in the second (Group 6), the usual embryo recovery time was followed. Recovery rate was higher in the former (89% vs. 31%; P < 0.01) than in the latter. After 4 days of culture, 53% of embryos reached compact morula or early blastocyst stage (16/30). Lambing rate was 57% for mouflon embryos transferred immediately and 56% for those cultured in vitro for 4 days; the lambing rate in the sheep control group was 71%. The length of gestation was longer in ewes carrying mouflons than in those carrying lambs (155 vs. 148 days).     

Horn growth related to testosterone secretion in two wild Mediterranean ruminant species: the Spanish ibex (Capra pyrenaica hispanica) and European mouflon (Ovis orientalis musimon)

Seasonal variations in the horn development and testicular activity of the Spanish ibex (Capra pyrenaica hispanica) (n=6) and European mouflon (Ovis orientalis musimon) (n=5) were monitored to determine the role of increasing testosterone concentration on the arrest of horn growth during the rutting season. Marked seasonal variations in the rate of horn growth (P<0.01) and testicular activity (P<0.001) were seen in both species, although the magnitude and timing of these changes were different (P<0.01). Horn growth rate was inversely correlated to seasonal levels in testosterone plasma concentration in both species (ibex: R=-0.45, P<0.01; mouflon R=-0.51, P<0.01). In the mouflon, the increase in plasma testosterone concentration recorded in September (P<0.05 compared with the lowest concentration) coincided with a significant reduction in horn growth (P<0.05). In the ibex, the increase in plasma testosterone concentration in October (P<0.05 compared with the lowest concentration) was associated with a significant arrest of horn growth in November (P<0.05). These results appear to support the hypothesis that high peripheral plasma levels of testosterone are linked with the seasonal arrest of horn growth during the rutting period.     

Ultrastructure of the cyst and life cycle of Sarcocystis sp. from wild sheep (Ovis musimon)

Sarcocystis sp. (Eimeriina: Sarcocystidae) is described as a heteroxenous coccidian with domestic dogs as an experimental definitive host and wild sheep (Ovis musimon) as natural intermediate hosts. Mature sarcocysts of this Sarcocystis sp. were examined by transmission electron microscopy. Sarcocysts in various muscle tissues were microscopic, had a thin primary cyst wall and septa and measured 81.0 x 30.5 microns. The cysts were located within muscle cells and were limited by a primary cyst wall (PCW). The cyst surface was highly folded forming densely packed projections. Between the PCW projections the surface of the cyst was marked with pit-like invaginations. The ground substance of the cyst formed a layer at the periphery of the cyst, filled the projections and formed septa which divided the cyst into compartments. Sarcocysts contained numerous bradyzoites that were 15.2 x 3 microns and few metrocytes 11.5 x 3.5 microns. Twelve days after ingesting Sarcocystis sp.-infected wild sheep meat, four dogs began passing sporocysts in their feces: two domestic cats did not pass oocysts or sporocysts after ingesting meat from the same animals. Sporocysts measured 14.8 x 9.9 microns.     

Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction

Aims: A new real‐time polymerase chain reaction‐based method was developed for the detection of Salmonella enterica in food. Methods and Results: The method consisted of a novel two‐step enrichment involving overnight incubation in buffered peptone water and a 5‐h subculture in Rappaport–Vassiliadis medium, lysis of bacterial cells and a Salmonella‐specific 5′‐nuclease real‐time PCR with an exogenous internal amplification control. Because a two‐step enrichment was used, the detection limit for dead S. enterica cells in artificially contaminated ice cream and salami samples was high at 10⁷ CFU (25 g)^?1, eliminating potential false‐positive results. When the method was evaluated with a range of 100 naturally contaminated food samples, three positive samples were detected by both the real‐time PCR‐based method and by the standard microbiological method, according to EN ISO 6579. When the real‐time PCR‐based method was evaluated alongside the standard microbiological method according to EN ISO 6579 with 36 food samples artificially contaminated at a level of 10⁰ CFU (25 g)^?1, identical results were obtained from both methods. Conclusions: The real‐time PCR‐based method involving a two‐step enrichment produced equivalent results to EN ISO 6579 on the day after sample receipt. Significance and Impact of the Study: The developed method is suitable for rapid detection of S. enterica in food.     

Genetic variability and individual assignment of Chinese indigenous sheep populations (Ovis aries) using microsatellites

The purpose of this study was to assess the genetic characteristics of six breeds of Chinese local sheep using 19 microsatellite loci and to effectively validate statistical methods for individual assignment based on informative microsatellites. All the six breeds deviated from Hardy–Weinberg equilibrium expectations, while the majority of markers complied. The polymorphism information content (PIC) of overall loci for the six populations ranged from 0.283 (SRCRSP5) to 0.852 (OarVH72). Tibetan sheep were the most diverse population with the highest mean allelic richness (6.895), while Ujmuqin (UQ) harboured the lowest allelic richness (6.000). The F‐statistics for the six populations were F_IS = ?0.172, F_IT = ?0.082 and F_ST = 0.077, respectively. Furthermore, the pair‐wise F_IS revealed a moderate genetic differentiation among populations (P < 0.01), indicating that all breeds can be considered genetically independent entities. The lowest genetic differentiation was between Tengchong (TC) and UQ (F_ST = 0.041), and the highest one was between TC and Fat‐tailed Han (F_ST = 0.111). In comparing the three statistical models, we note that the seven microsatellite loci (MAF65, OarJMP58, SRCRSP9, MCM140, OarAE129, BM8125 and SRCRSP5) commonly used for individual assignment will ensure a powerful detection of individual origin, with accuracy up to 91.87%, when the likelihood‐based method is used. Overall, these findings shed light onto the genetic characteristics of Chinese indigenous sheep and offer a set of microsatellite loci that is simple, economic and highly informative for individual assignment of Chinese sheep.     

Detection of plasmid DNA from all Chlamydia trachomatis serovars with a two-step polymerase chain reaction.

A polymerase chain reaction was used to amplify a 137-base-pair sequence of DNA from a Chlamydia trachomatis plasmid. Various parameters of the polymerase chain reaction were explored, and it was found that two short steps per reaction cycle were sufficient to achieve 10(12)-fold amplification in less than 1 h. By use of this procedure, 10(-18) g of a sequence of plasmid DNA, representing the amount of that sequence found in one C. trachomatis bacterium, was amplified to the point where it was clearly visible on an ethidium bromide-stained polyacrylamide gel under UV light. DNA from intact cells from each of the 15 serovars of C. trachomatis could also be amplified for visualization. With this procedure, the presence or absence of C. trachomatis DNA in a sample could be established in less than 1.5 h. The speed and extreme sensitivity of this detection procedure may make it a useful method for the detection of C. trachomatis, and similar techniques should be possible for any type of bacteria.     

Detection of hepatopancreatic parvovirus (HPV) in wild shrimp from India by nested polymerase chain reaction (PCR)

The prevalence of hepatopancreatic parvovirus (HPV) in wild penaeid shrimp samples from India was studied by nested polymerase chain reaction (PCR) using primers designed in our laboratory. The virus could be detected in 9 out of 119 samples by non-nested PCR. However, by nested PCR 69 out of 119 samples were positive. The PCR results were confirmed by hybridization with digoxigenin-labelled DNA probe. Shrimp species positive by non-nested PCR included Penaeus monodon, Penaeus indicus and Penaeus semisulcatus and by nested PCR Parapenaeopsis stylifera, Penaeus japonicus, Metapenaeus monoceros, M. affinis, M. elegans, M. dobsoni, M. ensis and Solenocera choprai. This is the first report on the prevalence of HPV in captured wild shrimp from India.     

Identification of trypanosomes in wild animals from southern Cameroon using the polymerase chain reaction (PCR)

One possible explanation of the maintenance of many historical foci of sleeping sickness in Central Africa could be the existence of a wild animal reservoir. In this study, PCR was used to detect the different trypanosome species present in wild animal captured by hunters in the southern forest belt of Cameroon (Bipindi). Trypanosomes were also detected by a parasitological method (Quantitative buffy coat: QBC). Parasite could not be isolated in culture medium (Kit for in vitro isolation: KIVI). Specific primers of T. brucei s.l., T. congolense forest type, T. congolense savannah type, T. vivax, T. simiae and T. b. gambiense group 1 were used to identify parasites in the blood of 164 animals belonging to 24 different species including ungulates, rodents, pangolins, carnivores, reptiles and primates. Of the 24 studied species, eight were carrying T. b. gambiense group 1. Those parasites pathogenic to man were found in monkeys (Cercocebus torquatus and Cercopithecus nictitans), in ungulates (Cephalophus dorsalis and C. monticola), in carnivores (Nandinia binotata and Genetta servalina) and in rodents (Cricetomys gambianus and Atherurus africanus). 13 species (54%) were carrying T. brucei s.l. identified as non-gambiense group 1.     

Establishment of pregnancy after the transfer of nuclear transfer embryos produced from the fusion of argali (Ovis ammon) nuclei into domestic sheep (Ovis aries) enucleated oocytes

Cloning mammalian species from cell lines of adult animals has been demonstrated. Aside from its importance for cloning multiple copies of genetically valuable livestock, cloning now has the potential to salvage endangered or even extinct species. The aim of this study was to investigate the effect of the bovine and domestic (Ovis aries) ovine oocyte cytoplasm on the nucleus of an established cell line from an endangered argali wild sheep (Ovis ammon) after nuclear transplantation. A fibroblast cell line was established from skin biopsies from an adult argali ram from the People's Republic of China. Early karyotype analysis of cells between 3-6 passages revealed a normal diploid chromosome number of 56. The argali karyotype consisted of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric and minute biarmed Y. Bovine ovaries were collected from a local abattoir, oocytes aspirated, and immediately placed in maturation medium consisting of M-199 containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 microg/mL streptomycin, 0.5 microg/mL follicle-stimulating hormone (FSH), 5.0 microg/mL luetinizing hormone (LH) and 1.0 microg/mL estradiol. Ovine (O. aries) oocytes were collected at surgery 25 hours postonset of estrus from the oviducts of superovulated donor animals. All cultures were carried out at 39 degrees C in a humidified atmosphere of 5% CO2 and air. In vitro matured MII bovine oocytes were enucleated 16-20 hours after onset of maturation and ovine oocytes within 2-3 hours after collection. Enucleation was confirmed using Hoechst 33342 and UV light. The donor argali cells were synchronized in G0-G1 phase by culturing in Dulbecco's modified Eagle's medium (DMEM) plus 0.5% fetal bovine serum for 5-10 days. Fusion of nuclear donor cell to an enucleated oocyte (cytoplast) to produce nuclear transfer (NT) embryos was induced by 2 electric pulses of 1.4 kV/cm for 30 microsc. Fused NT embryos were activated after 24 hours of maturation by exposure to ionomycin (5 microM, 4 minutes) followed by incubation in 6-dimethylaminopurine (0.2 mM, 4 hours) and cultured in microdrops of CR1aa medium. From a total of 166 constructed nuclear donor cell-bovine cytoplasm NT couples, 128 (77%) successfully fused, 100 (78%) developed to 8-16 cell stage, and 2 (1.56%) developed to the blastocyst stage. The presence of argali nuclei in 8-16 cell stage embryo clones was confirmed after observation of Hoechst 33342 stained embryos under UV light and chromosome analysis of metaphase spreads from blastomeres. A total of 127 constructed nuclear donor cell-ovine cytoplasm NT couples were produced, 101 (80%) successfully fused, 81 (80% of fused) developed to the 16- to 32-cell stage. A total of 28 hybrid (argali-sheep) and 21 sheep-sheep NT embryos were transferred into 6 recipients and 4 recipients, respectively. Two of these recipients, 1 carrying argali-sheep and 1 sheep-sheep, were confirmed pregnant at 49 days by ultrasound, but both pregnancies terminated by 59 days. The results of this study demonstrate the possibility of using xenogenic oocytes to produce early-stage embryos and pregnancies from an established fibroblast cell line of an endangered species.     

Mitochondrial DNA and Y-chromosomal diversity in ancient populations of domestic sheep (Ovis aries) in Finland: comparison with contemporary sheep breeds

Background

Several molecular and population genetic studies have focused on the native sheep breeds of Finland. In this work, we investigated their ancestral sheep populations from Iron Age, Medieval and Post-Medieval periods by sequencing a partial mitochondrial DNA D-loop and the 5’-promoter region of the SRY gene. We compared the maternal (mitochondrial DNA haplotypes) and paternal (SNP oY1) genetic diversity of ancient sheep in Finland with modern domestic sheep populations in Europe and Asia to study temporal changes in genetic variation and affinities between ancient and modern populations.

Results

A 523-bp mitochondrial DNA sequence was successfully amplified for 26 of 36 sheep ancient samples i.e. five, seven and 14 samples representative of Iron Age, Medieval and Post-Medieval sheep, respectively. Genetic diversity was analyzed within the cohorts. This ancient dataset was compared with present-day data consisting of 94 animals from 10 contemporary European breeds and with GenBank DNA sequence data to carry out a haplotype sharing analysis. Among the 18 ancient mitochondrial DNA haplotypes identified, 14 were present in the modern breeds. Ancient haplotypes were assigned to the highly divergent ovine haplogroups A and B, haplogroup B being the major lineage within the cohorts. Only two haplotypes were detected in the Iron Age samples, while the genetic diversity of the Medieval and Post-Medieval cohorts was higher. For three of the ancient DNA samples, Y-chromosome SRY gene sequences were amplified indicating that they originated from rams. The SRY gene of these three ancient ram samples contained SNP G-oY1, which is frequent in modern north-European sheep breeds.

Conclusions

Our study did not reveal any sign of major population replacement of native sheep in Finland since the Iron Age. Variations in the availability of archaeological remains may explain differences in genetic diversity estimates and patterns within the cohorts rather than demographic events that occurred in the past. Our ancient DNA results fit well with the genetic context of domestic sheep as determined by analyses of modern north-European sheep breeds.     

Distribution of nematode parasites of the digestive system in sheep (Ovis aries) and goats (Capra hircus) of the Piedmontese and Valdostano Alpine arc

A survey, carried out on gastro-intestinal nematodes of sheep and goats of Piemonte and of Valle d'Aosta (87 sheep and 12 goats) has shown the presence of the following species in sheep, Bunostomum trigonocephalum, Chabertia ovina, Cooperia curticei, Haemonchus contortus, Marshallagia marshalli, Nematodirus abnormalis, Nematodirus filicollis, Nematodirus helvetianus, Nematodirus spathiger, Oesophagostomum venulosum, Ostertagia circumcincta, Ostertagia lyrata, Ostertagia trifurcata, Skrjabinema ovis, Trichostrongylus axei, Trichostronglus colubriformis, Trichostronglus vitrinus, Trichuris ovis and Trichuris skrjabini; in goats, Bunostomum trigonocephalum, Chabertia ovina, Haemonchus contortus, Nematodirus filicollis, Nematodirus helvetianus, Oesophagostomum venulosum, Ostertagia circumcincta, Ostertagia ostertagi, Ostertagia trifurcata, Trichostrongylus axei, Trichostrongylus colubriformis and Trichostrongylus vitrinus. The percentage of each species in the two host is given in the text table.     

Gut region induces gastrointestinal microbiota community shift in Ujimqin sheep (Ovis aries): from a multi-domain perspective

Gastrointestinal (GI) microbiota is one of the most complicated microbial ecosystems and is vital in regulating biological processes associated with nutrient absorption and homeostatic maintenance. Although several efforts have been achieved in characterizing bacterial communities across gut regions, the variation of non-bacterial communities across GI tracts is still largely unexplored. To address this, we investigated microbial biogeography throughout the whole GI tracts of Ujimqin sheep (Ovis aries) by amplicon sequencing which targeted bacteria, fungi, and archaea. The results indicated that the community structures of all three domains were significantly distinguished according to GI tracts (stomach, small intestine, and large intestine), and a more strong and efficient species interaction was detected in small intestine based on cross-domain network analysis. Moreover, a between-domain difference in microbial assembly mechanism of among-GI regions was revealed here, wherein bacterial community is dominantly governed by variable selection (explaining ~62% of taxa turnover), while fungal and archaeal communities mainly governed by homogenizing dispersal (explaining ~49% and 60% of the turnover, respectively). Overall, these data highlight the GI section- and domain-dependence of GI microbial structure and assembly mechanism, suggesting that multi-domain should be explicitly considered when evaluating the influences of GI selection on gut microbial communities.