Single base editing system mediates site-directed mutagenesis of genes GDF9 and FecB in Ouler Tibetan sheep / 生物工程学报

In this study, a single base editing system was used to edit the FecB and GDF9 gene to achieve a targeted site mutation from A to G and from C to T in Ouler Tibetan sheep fibroblasts, and to test its editing efficiency. Firstly, we designed and synthesized sgRNA sequences targeting FecB and GDF9 genes of Ouler Tibetan sheep, followed by connection to epi-ABEmax and epi-BE4max plasmids to construct vectors and electrotransfer into Ouler Tibetan sheep fibroblasts. Finally, Sanger sequencing was performed to identify the target point mutation of FecB and GDF9 genes positive cells. T-A cloning was used to estimate the editing efficiency of the single base editing system. We obtained gRNA targeting FecB and GDF9 genes and constructed the vector aiming at mutating single base of FecB and GDF9 genes in Ouler Tibetan sheep. The editing efficiency for the target site of FecB gene was 39.13%, whereas the editing efficiency for the target sites (G260, G721 and G1184) of GDF9 gene were 10.52%, 26.67% and 8.00%, respectively. Achieving single base mutation in FecB and GDF9 genes may facilitate improving the reproduction traits of Ouler Tibetan sheep with multifetal lambs.

Meat quality of the Berrichon du Cher lambs and its cross-breeds with a native breed / Qualidade da carne dos cordeiros Berrichon du Cher e seus cruzamentos com raça nativa

This study evaluated the meat of lambs from crossbreeding of Polish lowland sheep, heather and rams of the Berrichone du Cher breed (B). The lambs were kept in a medium-intensive feeding system. During fattening, they received haylage and grass hay with the addition of clover, and a mixture of cereal grains with the addition of minerals and vitamins. During the fattening period, all feeds were given at will. The slaughter and cutting of the carcasses were performed in accordance with the technology applicable in the meat industry. In the longissimus lumborum muscle, the content of dry matter, total protein, total fat and ash was determined. In addition, the pH value of 45 minutes and 24 hours after slaughter, as well as the parameters of colour and tenderness of the meat were measured. The content of collagen protein, cholesterol and selected fatty acids was also determined. As a result of the conducted analyzes, the influence of the Berrichone du Cher breed on the improvement of quantitative parameters related to the slaughter value and some parameters related to the physicochemical composition of the hybrid meat was reported.

O objetivo deste estudo foi avaliar como o cruzamento da raça Berrichon du Cher com raças nativas de ovelhas polonesas de baixa altitude e ovelhas de charneca polonesa afeta os parâmetros qualitativos da carne .O maior teor de proteína (19,86 ± 0,36%, P <0,05) em cordeiros foi encontrado no grupo Berrichon du Cher e no grupo de cordeiros ovinos (19,23 ± 0,67%, P <0,05). Por outro lado, o maior teor de colágeno total (1,10 ± 0,21%) e a maior sensibilidade do tecido muscular (10,69 ± 0,74 N cm-2), que também apresentaram o menor diâmetro das fibras musculares (24,01 ± 4,62 µm, P < 0,01) foi encontrado nos cordeiros mestiços Berrichon du Cher e Polish Heath Sheep. Tanto a raça quanto o gênero afetaram significativamente o teor de colesterol na carne de cordeiro e o maior teor de colesterol foi encontrado na carne dos cordeiros Berrichon du Cher. Seu menor teor foi demonstrado na carne de Ovinos da Baixada Polonesa com a diferença de 9,1 mg 100g-1 de tecido muscular. Um dos níveis mais baixos do isômero CLA (C 18: 2 cis-9 trans-11) também foi encontrado no grupo dos cordeiros Berrichon du Cher. Este isômero evita a formação de colesterol. Dependência semelhante em relação ao gênero também foi observada. Os resultados obtidos indicam que as raças nativas são utilizadas em cruzamentos com a raça Berrichon du Cher destinada à engorda, principalmente para melhorar parâmetros quantitativos relacionados ao valor de abate e parâmetros selecionados de propriedades físicas e químicas da carne ovina.

Effect of dietary nutrition on tail fat deposition and evaluation of tail-related genes in fat-tailed sheep

BACKGROUND: The effects of dietary nutrition on tail fat deposition and the correlation between production performance and the Hh signaling pathway and OXCT1 were investigated in fat-tailed sheep. Tan sheep were fed different nutritional diets and the variances in tail length, width, thickness and tail weight as well as the mRNA expression of fat-related genes (C/EBPα, FAS, LPL, and HSL) were determined in the tail fat of sheep at three different growth stages based on their body weight. Furthermore, the correlations between tail phenotypes and the Hedgehog (Hh) signaling pathway components (IHH, PTCH1, SMO, and GLI1) and OXCT1 were investigated. RESULTS: C/EBPα, FAS, LPL, and HSL were expressed with differences in tail fat of sheep fed different nutritional diets at three different growth stages. The results of the two-way ANOVA showed the significant effect of nutrition, stage, and interaction on gene expression, except the between C/EBPα and growth stage. C/EBPα, FAS, and LPL were considerably correlated with the tail phenotypes. Furthermore, the results of the correlation analysis demonstrated a close relationship between the tail phenotypes and Hh signaling pathway and OXCT1. CONCLUSIONS: The present study demonstrated the gene-level role of dietary nutrition in promoting tail fat deposition and related tail fat-related genes. It provides a molecular basis by which nutritional balance and tail fat formation can be investigated and additional genes can be identified. The findings of the present study may help improve the production efficiency of fat-tailed sheep and identify crucial genes associated with tail fat deposition.

Addition of synthetic amino acids in sheep semen improves its quality after cryopreservation / Adição de aminoácidos sintéticos no sêmen ovino melhora a qualidade espermática após a criopreservação do sêmen

Some amino acids can protect mammalian sperm cells against oxidation during thermal stress caused by freezing/thawing. Thus, the objective was to evaluate the protective action of the association of the amino acids L-proline (Pro) and L-glutamine (Glu) against the cryoinjury caused to sheep sperm after cryopreservation. Eight ejaculates were collected from four sheep (n=32) and diluted in Tris-Egg Yolk-Glycerol until the final concentration of 200 x106 sptz/mL and kept in a water bath at 32 °C. The amino acids were added as follows: control (without adding amino acids), Pro+Glu 1 (100 µM Pro + 500 µM Glu), Pro+Glu 2 (300 µMPro + 1000 µM Glu), Pro+Glu 3 (500 µM Pro + 1500 µM Glu) and Pro+Glu 4 (700 µM Pro + 2000 µM Glu). Afterwards, the semen was cooled to 5 °C for 2 h, after that period, filled in 0.5 mL straws and then placed under liquid nitrogen vapor (N2L), 8 cm from the liquid sheet for 15min, and then immersed on the N2L. The samples were analyzed for sperm motility, plasma membrane and acrosomal membrane integrity, mitochondrial activity and binding test. The variables were subjected to the normality tests (Lilliefors test) and homoscedasticity tests (Cochran and Bartlett test), afterwards the variables of normal distribution were subjected to analysis of variance and the means compared by the Tukey test with a significance level of 5%. The Pro+Glu 3 group exhibited sperm with a greater (P<0.05) motility after thawing. In addition, the highest percentage of plasma and acrosomal membrane integrity were obtained using Pro+Glu 1, Pro+Glu 2 and Pro+Glu 3; and Pro+Glu 2 and Pro+Glu 3, respectively. Amino acids also kept mitochondrial activity high compared to the control, with Pro+Glu 3 resulting in greater activity (P<0.05). Sperm viability was higher (P<0.05) with the use of Pro+Glu 2 and Pro+Glu 3 than in the control. The number of sperm that showed the ability to bind to the egg yolk perivitelline membrane was higher (P<0.05) in semen treated with amino acids. It is concluded that the addition of synthetic amino acids in the semen of sheep before cryopreservation improves sperm quality and fertilization potential and can thus be added in cryopreservation protocols.

Alguns aminoácidos podem proteger as células espermáticas de mamíferos contra a oxidação durante o estresse térmico causado na congelação/descongelação. Dessa forma, objetivou-se avaliar a ação protetora da associação dos aminoácidos L-prolina (Pro) e L-glutamina (Glu) contra as crioinjúrias causadas aos espermatozoides de ovino após a criopreservação. Foram coletados oito ejaculados de quatro carneiros (n=32) e diluídos em Tris-Gema de ovo-Glicerol até a concentração final de 200 x106 sptz/mL e, mantidos em banho maria a 32 °C. Os aminoácidos foram adicionados da seguinte forma: controle (sem adição de aminoácidos), Pro+Glu 1 (100 µM Pro + 500 µM Glu), Pro+Glu 2 (300 µM Pro + 1000 µM Glu), Pro+Glu 3 (500 µM Pro + 1500 µM Glu) e Pro+Glu 4 (700 µM Pro + 2000 µM Glu). Depois, o sêmen foi resfriado a 5 °C por 2 h, após esse período, envasado em palhetas de 0,5 mL e então acondicionado sob vapor de nitrogênio líquido (N2L), a 8 cm da lâmina líquida por 15 min, e depois imersos no N2L. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática e da membrana acrossomal, atividade mitocondrial e teste de ligação. As variáveis foram submetidas aos testes de normalidade (Teste de Lilliefors) e homocedacidade (Teste de Cochran e Bartlett), posteriormente as variáveis de distribuição normal foram submetidas à análise de variância e as médias comparadas pelo teste de Tukey com nível de significância de 5%. O grupo Pro+Glu 3 exibiu espermatozoides com uma maior (P<0,05) motilidade após o descongelamento. Além disso o maior percentual de integridade da membrana plasmatica e acrossomal foram obtidos utilizando Pro+Glu 1, Pro+Glu 2 e Pro+Glu 3; e Pro+Glu 2 e Pro+Glu 3, respectivamente. Os aminoácidos também mantiveram alta a atividade mitocondrial em comparação com o controle, com Pro+Glu 3 resultando numa maior atividade (P<0,05). A viabilidade dos espermatozoides foi maior (P<0,05) com o uso de Pro+Glu 2 e Pro+Glu 3 do que no controle. O número de espermatozoides que apresentaram à capacidade de ligação a membrana perivitelina da gema de ovo foi maior (P<0,05) no sêmen tratado com aminoácidos. Conclui-se que, a adição dos aminoácidos sintéticos no sêmen de ovinos antes da criopreservação melhora a qualidade espermática e o potencial fecundante, podendo assim serem adicionados em protocolos de criopreservação.

Identification of single nucleotide polymorphisms in the prion protein gene in Santa Ines and Dorset sheep / Identificação de polimorfismos de nucleotídeos únicos em ovinos Santa Inês e Dorset através do gene da proteína priônica

Scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats and results from accumulation of the abnormal isoform of a prion protein in the central nervous system. Resistance or susceptibility to the disease is dependent on several factors, including the strain of infecting agent, the degree of exposure, and the presence of single nucleotide polymorphisms (SNPs) in the prion protein gene. The most important polymorphisms are present in codons 136, 154, and 171. SNPs have also been identified in other codons, such as 118, 127, 141, 142, and 143. The objective of this study was to investigate the genotypic profile of Santa Ines (n=94) and Dorset (n=69) sheep and identify polymorphisms in the prion protein gene using real-time PCR techniques and sequencing. We analyzed SNPs in 10 different codons (127, 136, 138, 140, 141, 142, 143, 154, 171, and 172) in Santa Ines sheep. Classification of the flock into risk groups associated with scrapie revealed that approximately 68% of the Santa Ines herd was considered at moderate risk (group 3), and the most frequent haplotype was ARQ/ARQ (47.8%). For Dorset sheep, 42% of the herd was considered at moderate risk (group 3), 40% at low risk (group 2), and 12% at very low risk (group 1). These findings improve our understanding of the genotype breed and further highlight the importance of genotyping and identification of polymorphisms in Brazilian herds to assess their effects on potential infections upon exposure to the sheep prion.(AU)

Scrapie é uma encefalopatia espongiforme transmissível que afeta ovinos e caprinos, resultante do acúmulo de uma isoforma anormal da proteína priônica no sistema nervoso central. A resistência ou susceptibilidade está relacionada a diversos fatores, tais como, a cepa do agente infectante, o grau de exposição e o polimorfismo de nucleotídeo único (SNPs) do gene da proteína priônica. Os principais polimorfismos estão presentes nos códons 136, 154 e 171. SNPs também são identificadas em outros códons, tais como, 118, 127, 141, 142, e 143. O objetivo do trabalho foi descrever o perfil genotípico de um rebanho da raça Santa Inês (n=94) e um rebanho da raça Dorset (n=89) para identificar potenciais polimorfismos através da técnica de PCR em tempo real e sequenciamento. Os achados no rebanho Santa Inês indicaram a presença de polimorfismos de nucleotídeos únicos em 10 códons diferentes (127, 136, 138, 140, 141, 142, 143, 154, 171 e 172). A classificação do rebanho, quanto aos grupos de risco associados ao scrapie, relevaram que aproximadamente 68% dos ovinos foram considerados do grupo de risco moderado (grupo 3), onde o haplótipo mais frequente foi ARQ/ARQ (47,8%). Para os ovinos da raça Dorset, 42% do rebanho foi considerado do grupo de risco moderado (grupo 3), 40% do grupo de risco baixo (grupo 2) e 12% do grupo de risco muito baixo. Os dados encontrados contribuem para o conhecimento do genótipo das raças, destacando a importância de trabalhos que relatam os polimorfismos genéticos para a identificação de rebanhos brasileiros, bem como o seu impacto a infecções com exposição ao príon ovino.(AU)

Survey of mutations in prolificacy genes in Santa Ines and Morada Nova sheep / Pesquisa de mutações em genes da prolificidade em ovelhas Santa Inês e Morada Nova

Polymorphisms in the BMP-15 gene related to Galway (FecXG) and Inverdale (FecXI) and in the BMPR-1B gene known as Booroola (FecB) mutations were investigated using the Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) method, on sheep from the breeds Santa Inês (n= 574) and Morada Nova (n=282). DNA was extracted and amplified through PCR with specific primers that introduced a restriction site in association with the mutation. The PCR products were submitted to endonucleases. The experiment found no FecXG and FecXI mutations. Six samples of animals with multiple offspring/birth history presented polymorphism for FecB similar to control samples, but this pattern was not confirmed by nucleotide sequencing. Although the absence of these mutations in the studied breeds, other factors related to prolificacy should be investigated to explain the inherent prolificity mechanisms.(AU)

Polimorfismos Galway (Fec XG ) e Inverdale (Fec XI), relacionados ao gene BMP-15, e Booroola (FecB), localizado no gene BMPR-1B, foram investigados usando-se a técnica de reação em cadeia da polimerase - polimorfismo de comprimento de fragmentos de restrição (PCR-RFLP), em ovelhas Santa Inês (n= 574) e Morada Nova (n=282). O DNA foi extraído e amplificado por PCR com iniciadores específicos, que introduziram um sítio de restrição associado à mutação, em seguida os amplicons foram submetidos à ação de endonucleases. Não foram observadas as mutações Fec XG e Fec XI nas amostras estudadas. Amostras de seis animais com histórico de partos gemelares apresentaram polimorfismo para FecB semelhantes às amostras controle, mas esse padrão não foi confirmado pelo sequenciamento de nucleotídeos. Apesar da ausência dessas mutações nos animais das raças estudadas, outros fatores relacionados à prolificidade devem ser pesquisados para explicar os mecanismos da alta prolificidade desses animais.(AU)

Productive response of hair ewes crossed with Damara and Dorper and growth of their lambs / Resposta produtiva de ovelhas de cabelo cruzado com Damara e Dorper e crescimento de seus cordeiros

Profitability of sheep production depends on the reproductive response of ewes and growth of their lambs, which can be improved through the ram genotype. The aim of this study was to evaluate the reproductive response of Blackbelly (Bb), Pelibuey (Pb), Katahdin (Kat) and Dorper (Dor) ewes crossed with Damara (Dam) and Dor rams and the growth of their offspring. To measure percentage of single and multiple lambings (MLP), 234 Bb, Pb, Kat and Dor ewes were used. To measure lamb growth, the offspring of 86 Bb x Pb (BbPb) ewes and 73 Kat x Dor (KatDOR) ewes were used. Four Dor x BbPb, six Dam x BbPb and six Dam x KatDor lambs were slaughtered for carcass assessment. MLP was analyzed with the CATMOD procedure considering the factors age and breed. Lamb growth data were analyzed with the MIXED procedure, and those of carcass characteristics with the GLM procedure of SAS, using weight at slaughter as covariable. MLP was higher (P<0.05) in Bb than in Dor. The Dor x BbPb lambs were heavier (P<0.05) at birth than Dam x BbPb. Of the lambs from single and multiple births, the KatDor lambs were heavier (P<0.05) at birth than BbPb lambs. The female lambs of Dor rams had higher (P<0.05) weight gain after weaning than those of Dam rams. Carcass dressing, subcutaneous fat, and primal cut yield were not different (P>0.05) by effect of ram breed. It is concluded that Bb ewes are more prolific than Dor ewes; male lambs of Dam rams had post-weaning growth response and carcass yield similar to those of Dor rams, although the female lambs of Dor rams were heavier than those of Dam rams.(AU)

Rentabilidade da produção de ovinos depende da resposta reprodutiva de ovelhas e do crescimento de seus cordeiros, o que pode ser melhorado por meio do genótipo masculino. O objetivo deste estudo foi avaliar o desempenho reprodutivo de ovinos Blackbelly (Bb), Pelibuey (Pb), Katadin (Kat) e Dorper (Dor) e o crescimento de cordeiros com carneiros Damara (Dam) e Dor. Para medir a porcentagem de partos simples e múltiplas (MLP), foram utilizados 234 ovelhas Bb, PB, Kat e Dor. Para medir o crescimento dos cordeiros, foram utilizadas 86 ovelhas Bb x Pb (BbPb) e 73 Kat x Dor (KatDOR). Quatro cordeiros Dor x BbPb, seis Dam x BbPb e seis Dam x KatDor foram sacrificados para avaliação de carcaça. MLP foi analisada com o procedimento CATMOD, considerando-se fatores como idade e raça. Dados de crescimento do cordeiro foram analisados com o procedimento MIXED, e as de características de carcaça com o procedimento GLM do SAS, utilizando-se peso ao sacrifício como covariável. MLP foi maior (P<0.05) em Bb do que em Dor. Os cordeiros Dor x BbPb foram mais pesados (P<0.05) no nascimento do que Dam x BbPb. Em partos simples e múltiplos, os cordeiros de ovelhas KatDor foram mais pesados (P<0.05) no momento do nascimento de cordeiros BbPb. As cordeiras de carneiros Dor apresentaram maior (P<0.05) ganho de peso após o desmame do que as de carneiros Dam. O rendimento de carcaça, a espessura da gordura subcutânea e o rendimento de corte primal não foram diferentes por efeito da raça de carneiro. Conclui-se que as ovelhas Bb são mais prolíficas do que as ovelhas Dor; cordeiros de carneiros Damara tiveram resposta de crescimento pós-desmame e rendimento de carcaça semelhantes aos dos carneiros Dor, embora Dorper cordeiras fossem mais pesadas do que as de Damara.(AU)

Productive response of hair ewes crossed with Damara and Dorper and growth of their lambs / Resposta produtiva de ovelhas de cabelo cruzado com Damara e Dorper e crescimento de seus cordeiros

Profitability of sheep production depends on the reproductive response of ewes and growth of their lambs, which can be improved through the ram genotype. The aim of this study was to evaluate the reproductive response of Blackbelly (Bb), Pelibuey (Pb), Katahdin (Kat) and Dorper (Dor) ewes crossed with Damara (Dam) and Dor rams and the growth of their offspring. To measure percentage of single and multiple lambings (MLP), 234 Bb, Pb, Kat and Dor ewes were used. To measure lamb growth, the offspring of 86 Bb x Pb (BbPb) ewes and 73 Kat x Dor (KatDOR) ewes were used. Four Dor x BbPb, six Dam x BbPb and six Dam x KatDor lambs were slaughtered for carcass assessment. MLP was analyzed with the CATMOD procedure considering the factors age and breed. Lamb growth data were analyzed with the MIXED procedure, and those of carcass characteristics with the GLM procedure of SAS, using weight at slaughter as covariable. MLP was higher (P0.05) by effect of ram breed. It is concluded that Bb ewes are more prolific than Dor ewes; male lambs of Dam rams had post-weaning growth response and carcass yield similar to those of Dor rams, although the female lambs of Dor rams were heavier than those of Dam rams.

Rentabilidade da produção de ovinos depende da resposta reprodutiva de ovelhas e do crescimento de seus cordeiros, o que pode ser melhorado por meio do genótipo masculino. O objetivo deste estudo foi avaliar o desempenho reprodutivo de ovinos Blackbelly (Bb), Pelibuey (Pb), Katadin (Kat) e Dorper (Dor) e o crescimento de cordeiros com carneiros Damara (Dam) e Dor. Para medir a porcentagem de partos simples e múltiplas (MLP), foram utilizados 234 ovelhas Bb, PB, Kat e Dor. Para medir o crescimento dos cordeiros, foram utilizadas 86 ovelhas Bb x Pb (BbPb) e 73 Kat x Dor (KatDOR). Quatro cordeiros Dor x BbPb, seis Dam x BbPb e seis Dam x KatDor foram sacrificados para avaliação de carcaça. MLP foi analisada com o procedimento CATMOD, considerando-se fatores como idade e raça. Dados de crescimento do cordeiro foram analisados com o procedimento MIXED, e as de características de carcaça com o procedimento GLM do SAS, utilizando-se peso ao sacrifício como covariável. MLP foi maior (P<0.05) em Bb do que em Dor. Os cordeiros Dor x BbPb foram mais pesados (P<0.05) no nascimento do que Dam x BbPb. Em partos simples e múltiplos, os cordeiros de ovelhas KatDor foram mais pesados (P<0.05) no momento do nascimento de cordeiros BbPb. As cordeiras de carneiros Dor apresentaram maior (P<0.05) ganho de peso após o desmame do que as de carneiros Dam. O rendimento de carcaça, a espessura da gordura subcutânea e o rendimento de corte primal não foram diferentes por efeito da raça de carneiro. Conclui-se que as ovelhas Bb são mais prolíficas do que as ovelhas Dor; cordeiros de carneiros Damara tiveram resposta de crescimento pós-desmame e rendimento de carcaça semelhantes aos dos carneiros Dor, embora Dorper cordeiras fossem mais pesadas do que as de Damara.

Molecular analysis of CIB4 gene and protein in Kermani sheep

The human calcium- and integrin-binding protein (CIB) family is composed of CIB1, CIB2, CIB3, and CIB4 proteins and the CIB4 gene affects fertility. Kermani sheep is one of the most important breeds of Iranian sheep breeds. The aim of this study was to analyze for the first time molecular characteristics of the CIB4 gene and protein in Kermani sheep. Different tissues were collected from the Kermani sheep and real time PCR was performed. The PCR products were sequenced, comparative analyses of the nucleotide sequences were performed, a phylogenetic tree was constructed, and different characteristics of CIB4 proteins were predicted. Real time PCR results showed that the CIB4 gene is expressed only in testis of Kermani sheep. The cDNA nucleotide sequence was identical with small tail Han sheep, cattle, goat, camel, horse, dog, mouse and human, respectively 100, 99, 99, 98, 98, 96, 96, and 96%. Hence, it can be suggested that the CIB4 gene plays a role in male fertility. Based on the phylogenetic analysis, sheep CIB4 gene has a close relationship with goat and cattle first, and then with camel and whale. Although we demonstrated that CIB4 is a testis-specific gene, expressed only in the testis and it interacts with other proteins, the mechanisms by which CIB4 expression is regulated need to be elucidated.

Identification of lamb flocks susceptible and resistant against Brachiaria poisoning / Identificação de rebanhos de cordeiros suscetíveis e resistentes a intoxicação por braquiária

This study was designed to assess the influence of genetic resistance against brachiaria poisoning in sheep. Two groups of sheep, one identified as susceptible (formed by two ewes and one ram) and the other as resistant against brachiaria poisoning (formed by three ewes and one ram) were selected. Sheep considered susceptible were those that presented clinical signs of brachiaria poisoning at any time of their life; resistant sheep were those that even raised on Brachiaria spp. pastures, did not developed any sign of the poisoning during their life. The offspring of the two flocks (15 lambs from the sensitive flock and 9 lambs from the resistant flock) were placed into brachiaria pasture (initially Brachiaria decumbens and B. brizantha,and only B. decumbens after weaning) and followed up during two years (2013-2014). The determination of protodioscin levels in B. decumbens pasture was performed only in 2014 and revealed significant amounts of the toxic principle. Eleven lambs of the susceptible group were affected to some degree of brachiaria poisoning and six died; no lamb of the resistant group was affected. Clinical signs consisted of varying degrees of subcutaneous edema of the face and, erythema and loss of hair of the ears, crusts on the skin of ears, around the eyes and on planum nasale, scar deformation of the ears, and bilateral ocular discharge; affected lambs also sought for shadowy shelters and they were poor doers. Several sheep recovered from the condition and then relapsed. Necropsy findings in six lambs included pale mucous membranes, emaciation, dermatitis, scar deformation of the ears, large yellow livers with marked lobular pattern, and moderate infestation by Haemonchus contortus...

Este estudo avaliou a resistência genética na ocorrência de intoxicação por braquiária em ovinos. Foram selecionados dois grupos de ovinos, um identificado como suscetível (formado por duas ovelhas e um carneiro) e o outro como resistente (formado por três ovelhas e um carneiro). Foram considerados suscetíveis ovinos que apresentaram sinais de intoxicação por Brachiaria spp. em algum ponto de suas vidas e resistentes aqueles ovinos que, mesmo criados em pastagem de braquiária, nunca desenvolveram qualquer sinal da intoxicação. A progênie desses dois grupos (15 cordeiros do grupo suscetível e 9 no grupo resistente) foi colocada numa pastagem de braquiária (inicialmente Brachiaria decumbens e B. brizantha e, após o desmame, apenas B. decumbens) e acompanhada durante dois anos (2013-2014). A determinação dos níveis de protodioscina em B. decumbens foi realizada apenas em 2014 e foram encontradas quantidades significativas do princípio tóxico. Onze cordeiros do grupo suscetível foram afetados por algum grau de intoxicação por braquiária; nenhum cordeiro do grupo resistente foi afetado. Os sinais clínicos consistiam de graus variáveis de edema subcutâneo da face e eritema e alopecia da pele das orelhas, crostas na pele das orelhas e ao redor dos olhos e no plano nasal, retração cicatricial das orelhas, fotofobia e corrimento ocular bilateral. Três cordeiros apresentaram desenvolvimento retardado. Vários cordeiros se recuperaram da condição, mas posteriormente quando foram colocados na pastagem apresentaram recidivas. Achados de necropsia em seis cordeiros incluíam mucosas pálidas, pobre condição corporal, dermatite, deformação cicatricial das orelhas, fígado aumentado de volume, amarelo e com padrão lobular evidenciado e graus moderados de infestação por Haemonchus contortus...

Polymorphism C242T in the Cyp19 gene in meat sheep / Polimorfismo no gene cyp19 em ovinos de corte

Abstract The objective of this study was to evaluate the frequency of C242T polymorphism on the aromatase gene and the allelic and genotypic frequency of these variants in sheep belonging to four breed groups. Blood samples were collected from 187 animals of four breed groups: Dorper, Santa Inês, Texel and White Dorper, originated from herds in the region of Maringá/PR, Brazil. The genomic DNA was extracted using alkaline extraction, with subsequent amplification of the fragments via PCR with specific primer. The samples resulting from amplification were subjected to digestion process using the Dpn II restriction enzyme and to polyacrylamide gel electrophoresis 10.0% and stained with silver nitrate. Three distinct genotypes were observed: homozygous (CC), heterozygous (CT) and homozygous for no cut (TT). The resulting data were analyzed using the POPGENE software with 5% significance. Genotypic frequencies among the breed groups were: Texel (CC - 0.426; CT - 0.511; TT - 0.064), Dorper (CC - 0.073; CT - 0.732; TT - 0.439), White Dorper (CC - 0.021; CT - 0.255; TT - 0.723) and Santa Inês (CC - 0.115; CT - 0.462; TT - 0.423).

Resumo O objetivo deste trabalho foi avaliar as frequências alélicas e genotípicas do polimorfismo do C242T no gene da aromatase em ovinos pertencentes a quatro grupos raciais. Foram coletadas amostras de sangue de 187 animais de quatro grupos raciais: Dorper, Santa Inês, Texel e White Dorper, provenientes de rebanhos da região de Maringá, PR - BR. O DNA genômico foi extraído utilizando o método de extração alcalina, com posterior amplificação dos fragmentos via PCR com primer específico. As amostras resultantes da amplificação foram submetidas ao processo de digestão com auxilio da enzima restrição Dpn II e submetido à eletroforese em gel de poliacrilamida de 10,0% e corado nitrato de prata. Foram observados três genótipos distintos: Homozigoto (CC), heterozigoto (CT) e homozigoto para não corte (TT). Os dados resultantes foram analisados utilizando o software POPGENE com significância de 5%. As frequências genotípicas entre os grupos raciais foram: Texel (CC - 0,426; CT - 0,511; TT - 0,064), Dorper (CC - 0,073; CT - 0,732; TT - 0,439), White Dorper (CC - 0,021; CT - 0,255; TT - 0,723) e Santa Inês (CC - 0,115; CT - 0,462; TT - 0,423).

Prevalence study of SNP c.421G>T in the ADAMTS2 gene responsible for dermatosparaxis in white dorper sheep in Brazil / Prevalência do SNP c.421G>T no gene ADAMTS2 responsável pela dermatosparaxia em ovinos white dorper no Brasil

Dermatosparaxis is an autosomal recessive disorder of connective tissue; the disorder is clinically characterized by skin fragility and hyperextensibility. Dermatosparaxis in White Dorper sheep is caused by a single nucleotide polymorphism (SNP) (c.421G>T) in the ADAM metalloproteinase with thrombospondin type 1 motif, 2 (ADAMTS2) gene. The aim of this study was to investigate the prevalence of this SNP in a White Dorper herd in São Paulo state, Brazil. In this study, we collected blood DNA samples from 303 White Dorper sheep and performed polymerase chain reaction to amplify the SNP region. The samples were sequenced to determine the presence of the SNP in the ADAMTS2 gene. The SNP prevalence in the studied population was 15.5%; this finding indicates that more effective control measures should be used to prevent the inheritance of SNP c.421G>T in the ADAMTS2 gene in Brazilian White Dorper herds.

A dermatosparaxia é uma doença autossômica recessiva do tecido conjuntivo, clinicamente caracaterizada pela fragilidade e hiperextensibilidade da pele. A dermatosparaxia em ovinos White Dorper é causada pelo polimorfismo de base única (SNP) c.421G>T no gene ADAM metalopeptidase com trombospondina tipo 1 motif, 2 (ADAMTS2). O objetivo deste estudo foi investigar a prevalência deste SNP em ovinos White Dorper no estado de São Paulo, Brasil. Foram coletadas amostras de sangue de 303 ovinos White Dorper. O DNA foi purificado destas amostras sanguíneas e utilizado em uma reação em cadeia da polimerase (PCR) para amplificação da região do gene contendo SNP c.421G>T. Os produtos das PCR foram sequenciados para determinar o genótipo dos animais. A prevalência do SNP na população estudada foi de 15,5%, estes achados indicam que medidas de controle efetivas devem ser utilizadas para prevenir a disseminação deste SNP no rebanho brasileiro de White Dorper.

Matrizes ovinas cruzadas são sempre mais eficientes do que as matrizes puras? / Are sheep crosses always more efficient than pure sheep?

Foi estudado o desempenho reprodutivo de 482 ovelhas multíparas (3/4 Texel x 1/4 Corriedale: TXC, 1/2 Romney Marsh x 1/2 Corriedale: RMC, 3/4 Poll Dorset x 1/4 Corriedale: PDC, 1/2 Finnish Landrace x 1/2 Merino Australiano: FLMA e Merino Australiano puras: MA), as características do crescimento de 585 cordeiros provenientes de carneiros Dorper e ovelhas: TXC (n = 68); RMC (n = 64); PDC (n = 68); FLMA (n = 183) e MA (n = 202) e os quilogramas de carne de cordeiros produzidos/kg de ovelha colocadas em cobertura (como indicador de eficiência), pastoreando campo natural e pastagens cultivadas. As ovelhas FLMA e TXC apresentaram as maiores taxas de parição (167,89 e 174,36%, respectivamente) em relação às demais cruzas, as quais não diferiram entre si (121,43 e 125,49%, PDC e RMC, respectivamente; P>0,05), porém foram superiores as MA (88,99%), sendo esse resultado uma consequência da maior quantidade de cordeiros nascidos/ovelha parida, que influenciou mais do que sua maior taxa de fertilidade. O genótipo da ovelha também afetou (P<0,0001) o crescimento dos cordeiros até o momento da sua venda com 41,6 ±6,7kg aos 206±7,25 dias de idade. Os cordeiros filhos das ovelhas TXC, PDC e RMC apresentaram maiores ganhos de peso diário que os filhos de FLMA, e esses maiores do que os MA. No entanto, de todas as cruzas avaliadas, somente a FLMA foi mais eficiente que a MA.

The reproductive performance of 482 multiparous ewes, with growth characteristics of 585 lambs from Dorper rams and ewes: 3/4 Texel x 1/4 Corriedale (TXC, n = 68); 1/2 Romney Marsh x 1/2 Corriedale (RMC, n = 64); 3/4 Poll Dorset x 1/4 Corriedale (PDC, n = 68); 1/2 Finnish Landrace x Australian Merino (FLAM, n = 183) and Australian Merino pure ewes (AM, n = 202) and kg of lamb produced/kg of sheep used in the service (as an indicator of efficiency), grazing natural and sown pastures, were studied. The TXC and FLAM sheep had the highest lambing percentage (167.89 and 174.36%, respectively) compared to the rest of the other crosses, and did not differ from each other (121.43 and 125.49%, PDC and RMC respectively, P>0.05), but were higher than AM (88.99%), due to the greater number of lambs born / ewe calving rather than their higher fertility. The type of ewe also affected (P<0.0001) lamb growth until its sale at 41.6±6.7 kg to 206±7.25 days of age, showing mothers of TXC lambs, PDC and RMC had higher daily gains than the FLAM and these more than those of MA. However, in all crosses tested, only FLAM was more efficient than MA.

Estimativas de parâmetros genéticos para características de crescimento em ovinos da raça Suffolk no Brasil / Estimates of genetic parameters for growth traits in Suffolk sheep in Brazil

O objetivo do trabalho foi estimar os parâmetros genéticos das características de crescimento da raça Suffolk, a fim de fornecer subsídio para a definição de estratégias de seleção para programas de melhoramento genético. Os dados analisados, coletados entre os anos de 2007 a 2009, são referentes a ovinos da raça Suffolk oriundos de uma propriedade localizada no Estado de São Paulo, Brasil, participante do programa de melhoramento genético Ovigol, conduzido pela empresa Aries Reprodução e Melhoramento Genético Ovino Ltda. em parceria com a empresa AbacusBio Limited, da Nova Zelândia. As características avaliadas foram: peso ao nascer (PN), ganho de peso pré-desmame (GPP) e peso ao desmame (PD), com número de registro de 1.039, 636 e 649 animais, respectivamente. Para análise estatística, utilizou-se o procedimento GLM do programa computacional SAS; para a consistência do pedigree e para as estimativas dos parâmetros genéticos, os programas computacionais utilizados foram Relax2: pedigree analysis program e o WOMBAT, respectivamente. O modelo linear geral incluiu sexo, grupo de contemporâneos (tipo de parto e ano de nascimento), covariável idade ao desmame (GPP e PD) com efeito quadrático, efeito genético aditivo direto, efeito de ambiente permanente materno e efeito residual. As herdabilidades para PN, GPP e PD foram 0,06, 0,42 e 0,37, respectivamente. As correlações genéticas entre PN e GPP, PN e PD e GPP e PD foram -0,10, -0,03 e 0,97, respectivamente. Na população estudada, as características GPP e PD, apresentam altas respostas à seleção, ao contrário do PN. As características PN e GPP são suficientes para compor um índice de seleção, sendo aconselhável o monitoramento do PN na fase inicial do programa.

The aim of this study was to estimate genetic parameters for growth of Suffolk, in order to provide a basis for the definition of selection strategies for breeding programs. The data analyzed are for Suffolk sheep, collected between years 2007 to 2009, from a property located in the State of São Paulo - Brazil, a participant in the Ovigol breeding program conducted by the company (Aries Reproduction and Breeding Sheep Ltd.) in partnership with (AbacusBio) New Zealand Limited. The evaluated characteristics were: birth weight (PN), weight gain pre-weaning (GPP) and weaning weight (PD), with registration number 1039, 636 and 649 respectively. For statistical analysis we used the GLM procedure of SAS software, for the consistency of pedigree and genetic parameter estimates software Relax2: pedigree analysis program and WOMBAT, respectively, were used. The general linear model included sex, contemporary group (type of birth and year of birth), covariate age at weaning (GPP and PD) with a quadratic effect, direct genetic effect, maternal permanent environmental effect and residual effect. Heritability estimates for PN, PD and GPP were 0.06, 0.42 and 0.37, respectively. Genetic correlations between PN and GPP, and PN and PD and GPP were -0.10, -0.03 and 0.97, respectively. In the study population characteristics GPP and PD have a high response to selection, unlike the PN. The GPP and PN characteristics are enough to make a selection index, it is advisable to monitor the PN in the initial phase of the program.

The power of 28 microsatellite markers for parentage testing in sheep

Background In sheep breeding, there are situations where relationships recorded at the farm among pedigrees such as parent-offspring, full-sibs or half-sibs need to be tested. A panel of 28 microsatellite (MST) markers was tested to provide accurate pedigree information and resolve the common problem of significant error in pedigree records in Merino sheep. Three different flocks of Australian Merino sheep were investigated. A private farm flock represents a flock with no record availability. Two other flocks were maintained under good managements of full keeping records and being selected for high and low parasite resistances. Results In the studied panel, eight MSTs provided an average of Polymorphic Information content (PIC) equal to 0.65 or more in order to be sufficient to make an accurate and successful DNA-based parentage analysis. The panel of twenty-eight MST loci was obviously sufficient for providing 100% accurate pedigree and genotyping data. DNA-based pedigree records were constructed and all significant pedigree record errors were eliminated. Conclusions These results were used for further study of population genetic parameters such as recombination and haplotyping which heavily based on pedigree information. Nevertheless MST based parentage testing is still available and affordable in most countries and for each farmer with reasonable cost in comparison with fast growing SNP based parentage technologies.

PPARgamma FAS, HSL mRNA and protein expression during Tan sheep fat-tail development

Background The objective of this study was to investigate proliferator-activated receptor (PPARγ), fatty acid synthase (FAS) and hormone-sensitive lipase (HSL) mRNA and protein expression in fat tails of Tan sheep. Rams from different developmental stages (aged 3, 6, 9, 12, 15 and 18 months) were selected, and their tail measurements including length (L), width (W) and girth (G) were recorded. The mRNA and protein expressions of PPARγ, FAS and HSL were examined by quantitative real-time polymerase chain reaction (PCR) and Western blot. Results The tail measurements increased with age. We observed no significant differences (P > 0.05) of PPARγ mRNA expression between ages 9 and 15 months, and between 12 and 15 months; FAS mRNA expression levels at each developmental stage were observed significantly in Tan sheep (P < 0.05); HSL mRNA expression with no significant differences were only observed between 6 and 15 months (P > 0.05). Significant differences (P < 0.05) of PPARγ, FAS and HSL protein expressions at each developmental stage were observed in Tan sheep. Conclusion We observed that the mRNA expression patterns of PPARγ and FAS decreased first before they increased again and then this process repeated. Conversely, the mRNA expression patterns of HSL increased first before they decreased and then this process repeated. The protein expression patterns of PPARγ and FAS decreased first before they increased again and then this process repeated. Conversely, the protein expression pattern of HSL increased first before it decreased again and then increased again.

Application of microsatellite markers for breeding and genetic conservation of herds of Pantaneiro sheep

Background The aim of the present study was to assess the genetic diversity of Pantaneiro sheep, using microsatellite markers, in order to assist maintenance and management plans, enhance mating systems and reduce the inbreeding rate. A total of 127 animals were genotyped at eight microsatellite loci. They belonged to populations from the Experimental Farm of the Universidade Federal da Grande Dourados (UFGD) (Dourados/MS/Brazil) and Embrapa Pantanal (Corumbá/MS/Brazil). Results The population of Pantaneiro sheep from the UFGD exhibited a high mean number of alleles (11.13) and allelic richness (10.66). The polymorphic information content was highly informative in the locus studied, resulting in a mean value of 0.71. Observed heterozygosity was lower than expected for all molecular markers assessed. The analysis of molecular variance showed a differentiation rate of 5.2% between populations. Conclusions The results of the statistical parameters indicated that populations of Pantaneiro sheep require special attention on herd management, and it's further necessary to implement breeder exchange programs in order to preserve the genetic variability of these populations. Furthermore, the maintenance of those populations in their typical habitats is rather required to allow different responses from the herds to the interactions between genotype and environment.

Características do sêmen de carneiros Dorper, Santa Inês e sem padrão racial definido, pré e pós-congelação, nos períodos chuvoso e seco / Characteristics of the semen of Dorper, Santa Ines and undefined breed sheep, pre-and post-freezing, in the rainy and dry period

Avaliaram-se as características espermáticas de carneiros Dorper, Santa Inês e sem padrão racial definido, nos períodos chuvoso e seco. Após ser colhido por vagina artificial, o sêmen foi avaliado quanto ao volume, ao turbilhonamento, à motilidade, ao vigor, à morfologia e à concentração, congelado e armazenado em botijão criogênico. Depois de descongelado, foram avaliadas a cinemática espermática, a integridade da membrana plasmática, a integridade do acrossoma e a atividade mitocondrial. Vigor espermático, motilidade total, motilidade progressiva, velocidade em linha reta e defeitos maiores não diferiram entre os períodos chuvoso e seco, porém volume, turbilhonamento, linearidade, retilinearidade e frequência de batimentos de cauda foram mais baixos (P<0,05) no período seco; já concentração espermática e defeitos totais apresentaram valores mais baixos no período chuvoso. Valores de integridade do acrossoma e da membrana plasmática, bem como o potencial de membrana mitocondrial, foram mais baixos (P<0,05) no período seco. Conclui-se que os períodos chuvoso e seco influenciam na qualidade espermática de ovinos criados na região Meio-Norte do Brasil e que esses animais têm uma qualidade espermática superior no período chuvoso, quando, portanto, deve ocorrer a criopreservação. Também se observou que, em relação à qualidade espermática, o melhor grupo de carneiros foi o Santa Inês...

The sperm characteristics of Dorper, Santa Ines and undefined breed ram in the rainy and dry seasons was assessed. After collection with an artificial vagina, the semen was evaluated for volume, turbulence, motility, viability, morphology and concentration, frozen and stored in a cryogenic cylinder. After thawing kinematic sperm, plasma membrane integrity, acrosome integrity and mitochondrial activity were evaluated. Sperm vigor, total motility, progressive motility, straight line speed and larger line defects did not differ between the rainy and dry seasons, however, volume, turbulence, linearity, straightness and frequency of tail beats were lower (P<0.05 ) in the dry period, and sperm concentration and total defects showed lower values during the rainy season. Values for acrosome integrity and plasma membrane and mitochondrial membrane potential were lower (P<0.05) in the dry season. It is concluded that the wet and dry period influence sperm quality in ram raised in the Mid - North region of Brazil, with a higher sperm quality in the rainy season, thus suggesting cryopreservation for that period. Also, the better ram group regarding sperm quality, was the Santa Ines...

Analysis of genetic diversity and differentiation of sheep populations in Jordan

Background Genetic diversity of sheep in Jordan was investigated using microsatellite markers (MS). Six ovine and bovine MS located on chromosomes 2 and 6 of sheep genome were genotyped on 294 individual from ten geographical regions. Results The number of alleles per locus (A), the expected heterozygosity (He) and observed heterozygosity (Ho) were measured. Overall A, He and Ho were 12.67, 0.820 and 0.684, respectively. On the other hand, genetic distances undoubtedly revealed the expected degree of differentiation among the studied populations. The finding showed closeness of three populations from south (Maan, Showbak and Tafeilah) to each other. Populations from the middle regions of Jordan (Karak, Madaba, Amman, AzZarqa and Mafraq) were found to be in one cluster. Only two populations of the middle region were an exception: AlSalt and Dead Sea. Finally, sheep populations from Irbid were located in separated cluster. It was clear that the studied predefined populations were subdivided from four populations and would be most probably accounted as ancestral populations. These results indicate that number of population is less than the predefined population as ten based on geographical sampling areas. Conclusions The possible inference might be that geographical location, genetic migration, similar selection forces, and common ancestor account for population admixture and subdivision of Awassi sheep breed in Jordan. Finally, the present study sheds new light on the molecular and population genetics of Awassi sheep from different regions of Jordan and to utilize the possible findings for future management of genetic conservation under conditions of climate changes and crossbreeding policy.

Scrapie e seu diagnóstico diferencial em ovinos no Mato Grosso do Sul / Scrapie and differential diagnosis in sheep in Mato Grosso do Sul, Brazil

Scrapie é uma doença infecciosa, neurodegenerativa fatal, causada pelo príon scrapie (PrPsc). Apresenta-se tanto na forma clássica em ovinos e caprinos geneticamente susceptíveis quanto na forma atípica em ovinos. A primeira notificação oficial do Brasil à Organização Mundial de Saúde Animal (OIE), um caso da forma clássica diagnosticado no Rio Grande do Sul ocorreu em 1985, mas a doença já havia sido diagnosticada no mesmo Estado em 1978. Este trabalho objetivou descrever dois surtos de Scrapie em ovinos em Mato Grosso do Sul (MS), Brasil e investigar, por meio de imuno-histoquímica (IHQ) a presença de PrPsc no Sistema Nervoso Central (SNC) de ovinos examinados entre 2003 e 2010. Na primeira parte observaram-se dois ovinos com sinais clínicos típicos de scrapie, detalhando-se os sinais neurológicos, dados epidemiológicos, histopatológicos e amostras teciduais em duplicata desses ovinos foram encaminhadas para realização de diagnóstico de Raiva e para diagnóstico IHQ para príon. Na segunda parte realizou-se levantamento de laudos de necropsia e diagnósticos histopatológicos de ovinos, no período de maio de 2003 a março de 2010. Amostras de sistema nervoso central de 51 casos foram selecionados, incluindo os dois já com diagnóstico de Scrapie mencionados acima; os tecido de todos esses ovinos foram submetidos à IHQ para detecção de proteína priônica. Os 49 ovinos avaliados apresentaram resultado negativo na IHQ para príon.

Scrapie is a fatal neurodegenerative infectious disease, caused by the scrapie prion (PrPsc), that can both in the as the classic form in genetically susceptible sheep and goats and in the atypical form in sheep. The first official notification of scrapie from Brazil was made to the World Organization for Animal Health (OIE) in 1985, in the state of Rio Grande do Sul, although the disease was first documented in this Brazilian state in 1978. The objective this paper was to describe two outbreaks of scrapie in sheep from Mato Grosso do Sul (MS), Brazil, and to investigate by immunohistochemistry (IHC) the presence of PrPsc in samples from the CNS of sheep examined during 2003 and 2010. The study was conducted in two stages; the first was the observation of two sheep with typical clinical signs of scrapie that underwent clinical examination with emphasis on neurological parameters, epidemiological data collection, necropsy and collection of samples in duplicate forwarded to the diagnosis of rabies, and for the IHC diagnosis of Transmissible Spongiform Encephalopathies. In the second part of the study, a survey was made the necropsy reviewing gross findings and histopathological diagnoses in sheep from May 2003 to March 2010. Samples of the central nervous system of fifty-one cases, including the two sheep mentioned above were subjected to IHC for detection of prion protein. The other 49 sheep, although displaying neurological-disease which should be included as scrapie differential diagnosis, had their tissues submitted to IHC resulting negative.