Functional Assessment of Diluent Choice for Semen Cryopreservation from Stallions with High and Low Freezability

Background: fertility rates using horse frozen-thawed semen remain lower than in other livestock species. This fact further suggests that horse semen hold intrinsic sensitivity to cryoinjury that must be investigated. Moreover, there is a substantial influence of genetic factors and diluent choice upon horse cryopreservation outcome. Collectively, these genetic and technical properties of horse semen could be explored to identify factors or conditions that may increase semen viability after freeze-thawing. The aim of this work was to evaluate the effect of diluents Botu-Crio®,Lactose-EDTA®, and INRA-82® on cryopreserved semen from stallions with high (HFA) and low freezability (LFA).Materials, Methods & Results: frozen-thawed semen was evaluated for motility, membrane integrity, and sperm DNA fragmentation using the thermoresistance test (TRT). Comparisons for each parameter were done in a pair-wise fashion between HFA and LFA semen at one-hour intervals during the TRT (0 h - 4 h). Sperm motility in HFA, regardless of the diluent, was larger (P < 0.05) than LFA, both on 0h and 1h. In the 2h evaluation, sperm motility using Botu-Crio® and Lactose-EDTA® was greater (P < 0.05) for HFA. Analysis of sperm membrane integrity was similar between HFA and LFA semen (P > 0.05) at 0 h and 3 h. Sperm DNA fragmentation was lower (P < 0.05) in HFA semen at 0 h and 1 h. Discussion: frozen-thawed semen from stallions of high freezing ability showed greater motility at all analysis, irrespectively of diluent choice, suggesting a strong influence of genetic factors on cryopreservation outcome. Membrane integrity was similar immediately after thawing but did differ later on other TRT time-points, irrespectively of diluent choice. As observed for motility, it was expected that sperm cells of stallions of HFA would show higher membrane integrity than their LFA counterparts. Sperm DNA fragmentation was quite low for both groups, as described in horses. Surprisingly, sperm DNA fragmentation incidence was constant throughout the analysis for both HFA and LFA. It was initially envisioned that increased DNA fragmentation would be found in semen from LFA stallions, since it is caused by multiple origins such as genetic factors. In conclusion, the semen diluent affects horse sperm motility after thawing, particularly from stallions with lower semen freezability

Influence of Male to Female Ratio on Hormone Profiles and Reproductive Performance of Anestrus Postpartum Ewes Subjected to the Male Effect

Background: Complete isolation of genders allows intense estrous induction and synchronization once rams are introduced in ewe flocks at the onset of the breeding season (BS). This management practice, defined as the male effect, results from a neuroendocrine process mediated by pheromones. The male effect is a straightforward procedure to induce estrous in noncycling ewes, but conditions for its use have not been fully explored. Thus, this study aimed to evaluate hormone levels and ovarian activity of postpartum ewes in anestrus which are subjected to the male effect under different male to female ratios.Material, Methods & Results: Pospartum females were selected according to body condition score and cyclicity status. Females were kept apart from males during 30 days at a distance of 10 m. Anestrus and ovulation were determined by P4 measures on days 10, 20 and 30 after isolation from males. After P4 concentration diagnosis, anestrus ewe (n = 99) were subjected to male to ewe ratios (MFR) of 1:20 (MFR20), 1:30 (MFR30) and 1:40 (MFR40). Santa Inês rams  (n = 3) of proven fertility were used. Three females of each group were randomly subjected to blood collection for LH concentration analysis. Ovarian activity was performed by ultrasonography after estrus manifestation in six ewe of each group. Estrus events were observed twice a day during the BS of 35 days, and estrus were considered synchronized when it occured within the initial five days of the BS. Pregnancy diagnosis was performed by ultrasonography on days 35 and 60 after the last mating. All ewe were in a non-cycling condition before BS onset, based upon P4 analysis. After initiation of the BS, P4 concentrations increased for all groups. Irrespectively of male to female ratio, male effect induced LH pre-ovulatory peaks within the initial 26 to 86 h of the BS. Synchronization of estrus reached 50% for MFR20, 40% for MFR30 and 20% for MFR40 for all ewe. Moreover, overall estrus incidence was 100% (MFR20), 90% (MFR30) and 65% (MFR40) within the initial 15 days of the BS. However, incidence of ewe that had repeated estrus events was lower for MFR20 than for MFR30. Follicular growth and number of ovulations was similar between groups. Conception rates on first service was higher than that of second service for MFR20 and MFR30, although there was no difference between services for MFR40. In contrast, overall conception rates, delivery type and prolificacy were similar between groups.Discussion: P4 increased to cyclicity levels after contact between genders, demonstrating the potential of the male effect to induce estrus in non-cycling ewes. Most ewe ovulated within three days after the male effect, possibly due to elevated basal LH levels. Moreover, the LH preovulatory peak varied within groups, possibly due to greater interactions between genders, which ultimately may have led to earlier ovulation anticipation under lower MFR. Estrus parameters were similar between groups, suggesting low or negligible effects of MFR. Ovulatory follicle size and growth and the number of ovulations were similar between all groups; previous reports have suggested that this may be due to a strong effect of their genetic background. Conception rates were higher at first than second services, demonstrating the potential of male effect. In conclusion, male to female ratio affects the efficiency of the male effect to induce and synchronize estrus in ewes under postpartum anestrus, but it does not affect conception rates and prolificacy

Lipid structural information from a single equine embryo by MALDI-MS

O objetivo deste trabalho foi relatar o potencial da técnica de MALDI-MS para caracterizar espécies de lipídios presentes em um único embrião equino e estudar algumas estruturas lipídicas detectadas por dissociação induzida por colisão (CID). No espectro de modo íon positivo, pudemos observar espécies, principalmente, protonadas e sodiadas de esfingomielinas (SM), fosfatidileolinas (PC) e triacilgliceróis (TAG). No modo negativo, observamos fosfatidiletanolaminas (PE) e fosfatidilinositos (PI). Espectros de íons de lípidos com maior intensidade foram utilizados para demonstrar o potencial da informação estrutural por MALDI-MS/MS. O espectro no modo positivo de m/z (massa sobre carga) 760,6 (atribuída como PC34:1) apresentou características de fragmentos PC de m/z 184,1 (denominada cabeça polar de colina), além de perda neutral (NL) de m/z 183 (fosforilcolina). Para o íon de m/z 766,6 (atribuída como PE38:5), observou-se a NL de 140, característica do PE. Para o íon de m/z 808,7 (38,5 atribuído como PC), além do fragmento m/z 184,1 na NL de 183, foi possível observar a perda de trimetilamina (íon de m/z 749,6) e o ciclofosfano (íon de m/z 147,0). Finalmente, para o modo de íon negativo, foram isolados e fragmentados o íon de m/z 863,6 que foi atribuído como PI36:1, devido à presença de m/z 153 (fosfato de glicerol – H2O-H ), 223 (inositol fosfo - 2H2O-H) , 241 (fosfoinositol – H2O-H), 281 (ácido oleico) e 581,3 (lisofosfoinositol – H2O+H). Concluímos que a MALDI - MS permite a detecção de uma ampla gama de espécies de PC, SM, PE, PI e TAG lipídicas, bem como a caracterização rápida e confiante de estruturas lipídicas a partir de um único embrião equino.The aim of this work was to report the potential of MALDI-MS for the characterization of lipid species present in a single equine embryo, and to study some lipid structures detected by collision induced dissociation (CID) experiments. In the positive ion mode spectrum, we could observe mostly protonated and sodiated species of sphingomyelins (SM), phosphatidylcholines (PC) and triacylglycerols (TAG). In the negative ion mode, we observed phosphatidylethanolamines (PE) and phosphatidylinositols (PI). MS/MS spectrum of most intense lipid ions was performed to show MALDI-MS/MS structural information potential. MS/MS spectrum in the positive mode of m/z 760.6 (attributed as PC34:1) depicted characteristic PC fragments of m/z 184.1 (choline polar head), and the neutral loss (NL) of 183 (phosphorylcholine). For the ion of m/z 766.6 (attributed as PE 38:5), we observed the NL of 140, characteristic of PE. For the ion of m/z 808.7 (attributed as PC 38.5), besides the fragment at m/z 184.1 at the NL of 183, it was possible to observe the loss of trimethylamine (ion of m/z 749.6), and the cyclophosphane (ion of m/z 147.0). Finally, for the negative ion mode, we isolated and fragmented the ion at m/z 863.6, which was attributed as PI 36:1 due to the presence of m/z 153 (glycerol phosphate – H2O-H), 223 (phospho inositol – 2H2O-H), 241 (phospho inositol – H2O-H), 281 (oleic acid), and 581.3 (lysophosphoinositol – H2O-H). We conclude that MALDI-MS allowed the detection of a broad range of PC, SM, PE, PI and TAG lipid species, as well as a fast and confident characterization of lipid structures from a single equine embryo.

Evaluation of clinical and reproductive parameters in Mangalarga Marchador mares treated with different doses of Cloprostenol or Dinoprost

Avaliou-se a ação de doses reduzidas e convencionais de substâncias luteolíticas sobre parâmetros clínicos e reprodutivos de éguas. As femeas receberam intramuscularmente, 125 ?g (n = 20) e 250 ?g (n = 20) de Cloprostenol e 2.5 mg (n = 20) e 5.0 mg (n = 20) de Dinoprost. A temperatura retal e as frequências cardíaca e respiratória foram aferidas antes e após a administração desses luteolíticos, considerando-se ainda a ocorrência de sudorese, diarreia, cólica e prostração. Monitorou-se o estro e o desenvolvimento folicular até a ovulação, quando realizou-se a inseminção artificial. A gestação foi diagnosticada com 300 e confirmada no 60o dia. Apenas as éguas tratadas com 2,5 e 5,0 mg de Dinoprost apresentaram alteração (P < 0.05) da frequência respiratória e os demais parâmetros não foram alterados (P > 0.05). A sudorese ocorreu em 5% e 10% das éguas tratadas, respectivamente, com 2.5 mg e 5.0 mg de Dinoprost e a diarréia em apenas 5% daquelas que receberam 5.0 mg desse luteolítico. As porcentagens de estro e prenhez das éguas tratadas com 125 ?g de Cloprostenol (45%/35%) e 2.5 mg de Dinoprost (50%/30%) foram menores (P < 0.05) do que os daquelas que receberam 250 ?g de Cloprostenol (85%/70%) e 5 mg de Dinoprost (90%/75%). O estro e a prenhez das éguas Controle foram menores (P < 0.05) do que nas tratadas. Conclui-se que apesar de não promoverem alterações significativas dos parâmetros clínicos, as doses reduzidas não apresentam as mesmas eficiências dos tratamentos com doses convencionais para induzir o estro

Efficient, fast and low-cost strategies for DNA extraction from different nucleated sheep cells

DNA extraction is usually the first step to perform molecular studies. This process can be nonviable due to genomic DNA (gDNA) extraction commercial kits prices. Furthermore, available DNA extraction protocols generally have high specificity, limiting their use to specific sources of biological material. In order to reduce costs, optimize time and laboratory logistics, besides to demonstrate a versatile protocol, the present study worked on an efficient DNA extraction protocol from somatic and non-somatic cells, using biological material from sheep as a model. For that, gDNA was extracted from whole blood, spermatozoa, and hair bulb cells, collected from three adult sheep, transported at 5ºC and stored at -20ºC until lab procedures. After extraction, gDNA concentration and purity were evaluated in a nano spectrophotometer. gDNA concentration from whole blood was greater (p < 0.05) than extracted from hair bulb cells, which in turn was superior (p < 0.05) than in spermatozoa. Also, gDNA from whole blood and, followed by, sperm showed greater (p < 0.05) purity when compared to gDNA of hair bulb cells. Adapting a gDNA extraction protocol, originally developed for bovine whole blood, enabled to obtain and isolate gDNA in different nucleated sheep cell

Efeito da exposição aos crioprotetores glicerol e metilformamida sobre a viabilidade e fertilidade do sêmen equino

O processo de congelação e descongelação de sêmen induz a diversos efeitos deletérios na célula espermática. Crioprotetores são adicionados aos meios de congelação para minimizar esses danos, entretanto altas concentrações dessas substâncias aumentam sua toxicidade e, conseqüentemente, diminui a fertilidade. É importante o equilíbrio entre habilidade crioprotetora e toxicidade desses componentes. As amidas têm se mostrado um crioprotetor de baixa toxicidade, especialmente para garanhões de má congelabilidade e para outras espécies de animais. O presente estudo objetivou determinar a viabilidade e o efeito contraceptivo dos crioprotetores glicerol (GL) e metilformamida (MF) para congelação de sêmen de garanhões. EXPERIMENTO I: Foi colhido o sêmen de 10 garanhões com uma vagina artificial, diluído (1:1) em meio à base de leite (Botu-sêmen®) e centrifugado (600xg/10min). Foram ressuspendidos os pellets com meio de congelação (INRA82) contendo três concentrações diferentes de GL (2, 3 e 4,5%) e MF (2, 3 e 4,5%) e um grupo controle (Contr.) sem adição de crioprotetor, na concentração de 100x106 espermatozóides/mL. O sêmen foi envasado em palhetas de 0,5mL, resfriado a 5°C/1hora e mantido em vapor de nitrogênio durante 15 minutos e daí imerso em nitrogênio líquido. As amostras foram avaliadas após o período de estabilização (5°C/1hora) bem como após a descongelação (46°C/20seg) pelo sistema computadorizado (HTMA-IVOS-10). Foram avaliados parâmetros de motilidade, e através de Microscopia de Fluorescência, foi avaliada a integridade de membranas (plasmática e acrossomal) e potencial da membrana mitocondrial. A análise estatística foi realizada por ANOVA e teste de Tukey com significância de 5%. No período pós-estabilização a única diferença observada foi no parâmetro de motilidade progressiva, onde o grupo MF2% (25,7%) foi superior...The frozen thawing semen process induce several damages on sperm cells. The use of cryoprotectors is fundamental to protect the spermatozoa from frozen-thawing damage, however high concentrations of these compounds can decrease the fertility. It is very important to balance the ability to cryoprotect with cryoprotector toxicity. Amidas has been shown to have a low toxicity specially for bad freezer stallions and some other species. The present experiment aimed to determine the effect of exposition on glycerol (GLY) and methylformamida (MF) over viability and fertility of stallion semen. On experiment I one ejaculated from 10 stallions was collected and diluted (1:1) in skim milk extender (Botu-sêmen®) and centrifuged (600g/10min). The sperm pellets were resuspended with a frozen extender (INRA82) containing 3 different concentrations of GLY (2, 3 or 4,5%) and MF (2, 3 or 4,5%) and a control group. (Contr.) using a concentration of de 100x106 esperm/mL. Semen was packaged in 0,5mL straws, cooled at a 5°C/1hour, maintained over nitrogen and finally frozen. The motility parameters were evaluated after stabilization (5°C/1hour) and again after frozen thawing (46°C/20seg) by CASA (HTMA-IVOS-10). Membrane integrity (Plasmatic and Acrossomal) and mitochondrial potential of membrane were also evaluated using fluorescent probes. ANOVA and Tukey test were used for statistical evaluation. After the stabilization time the only parameter that was different was the progressive motility (PM) that was higher on MF2% group (25,7%) when compared with Control Group (13,5%). No differences (p>0.05) were observed on Total Motility (TM) when concentrations of the same cryoprotector were compared, however differences favorable to MF were detected when comparisons were...(Complete abstract, click electronic access below

Influence of Male to Female Ratio on Hormone Profiles and Reproductive Performance of Anestrus Postpartum Ewes Subjected to the Male Effect

Background: Complete isolation of genders allows intense estrous induction and synchronization once rams are introduced in ewe flocks at the onset of the breeding season (BS). This management practice, defined as the male effect, results from a neuroendocrine process mediated by pheromones. The male effect is a straightforward procedure to induce estrous in noncycling ewes, but conditions for its use have not been fully explored. Thus, this study aimed to evaluate hormone levels and ovarian activity of postpartum ewes in anestrus which are subjected to the male effect under different male to female ratios.Material, Methods &amp; Results: Pospartum females were selected according to body condition score and cyclicity status. Females were kept apart from males during 30 days at a distance of 10 m. Anestrus and ovulation were determined by P4 measures on days 10, 20 and 30 after isolation from males. After P4 concentration diagnosis, anestrus ewe (n = 99) were subjected to male to ewe ratios (MFR) of 1:20 (MFR20), 1:30 (MFR30) and 1:40 (MFR40). Santa Inês rams  (n = 3) of proven fertility were used. Three females of each group were randomly subjected to blood collection for LH concentration analysis. Ovarian activity was performed by ultrasonography after estrus manifestation in six ewe of each group. Estrus events were observed twice a day during the BS of 35 days, and estrus were considered synchronized when it occured within the initial five days of the BS. Pregnancy diagnosis was performed by ultrasonography on days 35 and 60 after the last mating. All ewe were in a non-cycling condition before BS onset, based upon P4 analysis. After initiation of the BS, P4 concentrations increased for all groups. Irrespectively of male to female ratio, male effect induced LH pre-ovulatory peaks within the initial 26 to 86 h of the BS. Synchronization of estrus reached 50% for MFR20, 40% for MFR30 and 20% for MFR40 for all ewe. Moreover, overall estrus incidence was 100% (MFR20), 90% (MFR30) and 65% (MFR40) within the initial 15 days of the BS. However, incidence of ewe that had repeated estrus events was lower for MFR20 than for MFR30. Follicular growth and number of ovulations was similar between groups. Conception rates on first service was higher than that of second service for MFR20 and MFR30, although there was no difference between services for MFR40. In contrast, overall conception rates, delivery type and prolificacy were similar between groups.Discussion: P4 increased to cyclicity levels after contact between genders, demonstrating the potential of the male effect to induce estrus in non-cycling ewes. Most ewe ovulated within three days after the male effect, possibly due to elevated basal LH levels. Moreover, the LH preovulatory peak varied within groups, possibly due to greater interactions between genders, which ultimately may have led to earlier ovulation anticipation under lower MFR. Estrus parameters were similar between groups, suggesting low or negligible effects of MFR. Ovulatory follicle size and growth and the number of ovulations were similar between all groups; previous reports have suggested that this may be due to a strong effect of their genetic background. Conception rates were higher at first than second services, demonstrating the potential of male effect. In conclusion, male to female ratio affects the efficiency of the male effect to induce and synchronize estrus in ewes under postpartum anestrus, but it does not affect conception rates and prolificacy

Uso de soro autólogo como adjuvante no tratamento de úlcera de córnea em equino: Relato de caso

O presente trabalho relata o uso do soro autólogo como adjuvante no tratamento de úlcera de córnea de equinos. No dia 08/09/2018 foi atendido no Hospital Veterinário Dr. Vicente Borelli, um equino macho, Quarto de Milha, quatro anos de idade, pesando 460 kg, submetido à uma dieta diária composta de 5 kg de concentrado e volumoso e água ad libitum. O animal participou de prova equestre, transportado em um veículo fechado (caminhão). Assim que retornou à propriedade apresentou desconforto ocular. Solicitou-se o serviço veterinário, que confirmou o diagnóstico para úlcera de córnea e realizou tratamento que não obteve êxito. Na sequência, o animal foi encaminhando ao Hospital Veterinário. No exame clínico geral todos os parâmetros encontravam-se dentro normalidade. Procedeu-se exame clínico oftálmico, identificando blefaroespasmos, irregularidade e opacidade de córnea e câmara anterior, presença de ejeção ciliar, com teste positivo para fluoresceína. Foi iniciado um tratamento sistêmico com administração de Flunixin meglumine durante cinco dias. Dessa forma, para o tratamento tópico foi efetuada a confecção do soro autólogo, colhidos em tubos vermelhos com ativador de coágulos de 5 mL centrifugados à 3.000 rpm. Foram instiladas duas gotas de sulfato de atropina a 1%/BID nos dias 08/09 e 09/09. Entre os dias 08/09 e 14/09 foi instituído duas gotas de sulfato de tobramicina e soro autólogo a cada uma hora. Nos dias 15/09 e 16/09 a frequência do uso do sulfato de tobramicina e soro autólogo foi de duas horas, totalizando 12 aplicações por dia. Foi procedido um novo teste de fluoresceína notando-se uma redução de aproximadamente 70%. Do dia 17/09 até 20/09 a frequência foi alterada para seis horas entre as aplicações. O teste de fluoresceína foi refeito, o qual se mostrou negativo. No dia 21/09 cessou o uso do soro autólogo, mantendo as aplicações do sulfato de tobramicina até o dia 24/09. Ainda no dia 21/09 foram instiladas duas gotas de diclofenaco de sódio 0,1%, quatro vezes ao dia, até o dia 28/09/2018. Nesse mesmo dia o paciente obteve alta hospitalar. Conclui-se que o soro autólogo atua como adjuvante em úlceras de córnea em equino, conferindo resultados satisfatórios, apresentando baixo custo, praticidade de preparo e minimizando o emprego de tratamentos convencionais sem resposta clínica

Lipid structural information from a single equine embryo by MALDI-MS

O objetivo deste trabalho foi relatar o potencial da técnica de MALDI-MS para caracterizar espécies de lipídios presentes em um único embrião equino e estudar algumas estruturas lipídicas detectadas por dissociação induzida por colisão (CID). No espectro de modo íon positivo, pudemos observar espécies, principalmente, protonadas e sodiadas de esfingomielinas (SM), fosfatidileolinas (PC) e triacilgliceróis (TAG). No modo negativo, observamos fosfatidiletanolaminas (PE) e fosfatidilinositos (PI). Espectros de íons de lípidos com maior intensidade foram utilizados para demonstrar o potencial da informação estrutural por MALDI-MS/MS. O espectro no modo positivo de m/z (massa sobre carga) 760,6 (atribuída como PC34:1) apresentou características de fragmentos PC de m/z 184,1 (denominada cabeça polar de colina), além de perda neutral (NL) de m/z 183 (fosforilcolina). Para o íon de m/z 766,6 (atribuída como PE38:5), observou-se a NL de 140, característica do PE. Para o íon de m/z 808,7 (38,5 atribuído como PC), além do fragmento m/z 184,1 na NL de 183, foi possível observar a perda de trimetilamina (íon de m/z 749,6) e o ciclofosfano (íon de m/z 147,0). Finalmente, para o modo de íon negativo, foram isolados e fragmentados o íon de m/z 863,6 que foi atribuído como PI36:1, devido à presença de m/z 153 (fosfato de glicerol – H2O-H ), 223 (inositol fosfo - 2H2O-H) , 241 (fosfoinositol – H2O-H), 281 (ácido oleico) e 581,3 (lisofosfoinositol – H2O+H). Concluímos que a MALDI - MS permite a detecção de uma ampla gama de espécies de PC, SM, PE, PI e TAG lipídicas, bem como a caracterização rápida e confiante de estruturas lipídicas a partir de um único embrião equino