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BACKGROUND: Non-surgical artificial insemination techniques for sheep may benefit from larger numbers of sperm in the insemination dose because the ewe cervix is convoluted and often cannot be traversed with an insemination gun resulting in deposition of the sperm at the os cervix. OBJECTIVE: To compare a range of sperm concentrations when cryopreserving semen from Santa Ines rams and determine the effects of this on post-thaw quality. MATERIALS AND METHODS: One ejaculate from each ram (n = 10) was diluted to four sperm concentrations to obtain the following groups: G-400, G-800, G-1200, and G-1600 x 106 sperm/mL. The semen samples were packaged in 0.25 mL straws, cooled to 5 degree C, cryopreserved in liquid nitrogen vapor, thawed in a water bath (40 degree C per 20 s), and were analyzed for computerized kinetics, capacitation and acrosome integrity, and plasma membrane integrity of sperm. RESULTS: The G-400 treatment resulted in samples with the highest linearity and progressive motion (P < 0.05) and had significantly greater plasma membrane integrity, and lower capacitation and acrosome reaction rates compared to G-1600 (P < 0.05). Overall, use of the G-400 treatment resulted in better kinetics, less plasma membrane damage and less early capacitation. However, despite reducing the ejaculate yield and increasing the costs of the semen freezing process, the G-800 and G-1200 treatments make a greater absolute number of sperm with good kinetics, plasma membrane integrity and capacitation status available. CONCLUSION: Ram sperm concentration impacts cryopreservation, and higher concentrations may be advantageous if a single artificial insemination protocol is desirable. doi.org/10.54680/fr22610110812.

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The objective of this study was to quantify the superovulatory response and embryo production of Brazilian Bergamasca sheep and to evaluate the link to the follicular condition before superovulatory treatment, as a reference for selection of donors with potential for superovulation. Follicular population of twenty-three sheep was evaluated by ultrasound during metestrus phase of the estrous cycle and divided into groups of low, medium and high follicular population. Subsequently, they were synchronized, superovulated with 133mg of pFSH, mated and subjected to embryo collection. The superovulatory response (9.0±3.3 vs 10.7±6.2 vs 13.8±7.1) and embryo production (4.0±3.8 vs 2.6±2.0 vs 1,8±4.0) were similar between groups (P>0.05). There was a positive correlation between the number of follicles during the metestrus phase and the number of corpus luteum with premature regression (PLR) (0.52) and a negative correlation between the recovery rate and PLR (-0.44) (P<0.05). The sheep that presented PLR had more follicles during metestrus (16.9±7.8 vs 12.7±3.2) and lower embryo recovery rate (38.8±29.3 vs 72.2±29.9) than those with functional CLs (P<0.05). Follicular quantification during metestrus phases was unable to identify donors with high embryo production. Animals with PLR had a larger follicular population during metestrus and lower embryo recovery rate.(AU)

O objetivo deste trabalho foi quantificar a resposta superovulatória e a produção embrionária de ovelhas Bergamácia Brasileira e relacioná-las com a condição folicular antes do tratamento superovulatório, como referência para seleção de doadoras com potencial para superovulação. Vinte e três ovelhas foram avaliadas quanto à população folicular por ultrassonografia na fase de metaestro do ciclo estral e divididas em grupos com baixa, média e alta população folicular. Posteriormente foram sincronizadas, superovuladas com 133mg de pFSH, acasaladas e submetidas à coleta de embriões. A resposta superovulatória (9,0±3,3 vs. 10,7±6,2 vs. 13,8±7,1) e a produção embrionária (4,0±3,8 vs. 2,6±2,0 vs. 1,8±4,0) foram semelhantes entre os grupos (P>0,05). Houve correlação positiva entre o número de folículos no metaestro e o número de corpos lúteos com regressão prematura (RPCL) (0,52) e correlação negativa entre a taxa de recuperação e RPCL (-0,44) (P <0,05). As ovelhas que apresentaram RPCL tiveram mais folículos no metaestro (16,9±7,8 vs. 12,7±3,2) e menor taxa de recuperação embrionária (38,8±29,3 vs. 72,2±29,9) do que as que apresentaram CLs funcionais (P<0,05). A quantificação folicular nas fases de metaestro não foi capaz de identificar doadoras com alto potencial de produção embrionária. Animais com RPCL tiveram maior população folicular no metaestro e menor recuperação de embriões.(AU)

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Piau porcine blastocysts were submitted to MALDI-TOF to identify the main phospholipids (PL). After that, in vivo blastocysts (D6) were vitrified (n=52), non-vitrified were used as control (n=42). After warming, blastocysts were in vitro cultured to assess re-expansion and hatching at 24 and 48 hours. Finally, at 48 hours, hatched blastocysts were submitted to RT-qPCR searching for BCL2A1, BAK, BAX and CASP3 genes. For MALDI-TOF, the ion intensity was expressed in arbitrary units. Blastocyst development was compared by Qui-square (P< 0.05). Among the most representative PL was the phosphatidylcholine [PC (32:0) + H]+; [PC (34:1) + H]+ and [PC (36:4) + H]+. Beyond the PL, MALDI revealed some triglycerides (TG), including PPL (50:2) + Na+, PPO (50:1) + Na+, PLO (52:3) + Na+ and POO (52:2) + Na. Re-expansion did not differ (P> 0.05) between fresh or vitrified blastocysts at 24 (33.3%; 32.7%) or 48 hours (2.4%; 13.5%). Hatching rates were higher (P< 0.05) for fresh compared to vitrified at 24 (66.7%; 15.4%) and 48 hours (97.6%; 36.0%). BAX was overexpressed (P< 0.05) after vitrification. In conclusion, Piau blastocysts can be cryopreserved by Cryotop. This study also demonstrated that the apoptotic pathway may be responsible for the low efficiency of porcine embryo cryopreservation.(AU)

Blastocistos de suínos foram submetidos ao MALDI-TOF para se identificarem os principais fosfolipídios (PL). Depois, parte destes embriões (D6) foram vitrificados (n=52), ou permaneceram frescos (grupo controle, n=42). Após o aquecimento, os blastocistos foram cultivados in vitro para se avaliar a reexpansão e a eclosão (BE) às 24 e 48 horas. Finalmente, às 48 horas, os BE foram submetidos ao RT-qPCR em busca dos genes BCL2A1, BAK, BAX e CASP3. No MALDI-TOF, a intensidade do íon foi expressa em unidades arbitrárias. O desenvolvimento embrionário foi comparado por qui-quadrado (P<0,05). Entre os PL mais representativos estavam as fosfatidilcolinas [PC (32: 0) + H] +; [PC (34: 1) + H] + e [PC (36: 4) + H] +. Além do PL, o MALDI revelou alguns triglicerídeos (TG), incluindo PPL (50: 2) + Na +, PPO (50: 1) + Na +, PLO (52: 3) + Na + e POO (52: 2) + Na. A reexpansão não diferiu (P>0,05) entre blastocistos frescos ou vitrificados às 24 (33,3%, 32,7%) e 48 horas (2,4%, 13,5%). As taxas de eclosão foram maiores (P<0,05) para o grupo fresco comparado ao vitrificado às 24 (66,7% x 15,4%) e 48 horas (97,6% x 36,0%). O BAX estava mais expresso (P<0,05) após a vitrificação. Concluindo, os blastocistos Piau podem ser criopreservados por Cryotop. Este estudo também demonstrou que a via apoptótica pode ser responsável pela baixa eficiência da criopreservação de embriões suínos.(AU)

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Piau porcine blastocysts were submitted to MALDI-TOF to identify the main phospholipids (PL). After that, in vivo blastocysts (D6) were vitrified (n=52), non-vitrified were used as control (n=42). After warming, blastocysts were in vitro cultured to assess re-expansion and hatching at 24 and 48 hours. Finally, at 48 hours, hatched blastocysts were submitted to RT-qPCR searching for BCL2A1, BAK, BAX and CASP3 genes. For MALDI-TOF, the ion intensity was expressed in arbitrary units. Blastocyst development was compared by Qui-square (P< 0.05). Among the most representative PL was the phosphatidylcholine [PC (32:0) + H]+; [PC (34:1) + H]+ and [PC (36:4) + H]+. Beyond the PL, MALDI revealed some triglycerides (TG), including PPL (50:2) + Na+, PPO (50:1) + Na+, PLO (52:3) + Na+ and POO (52:2) + Na. Re-expansion did not differ (P> 0.05) between fresh or vitrified blastocysts at 24 (33.3%; 32.7%) or 48 hours (2.4%; 13.5%). Hatching rates were higher (P< 0.05) for fresh compared to vitrified at 24 (66.7%; 15.4%) and 48 hours (97.6%; 36.0%). BAX was overexpressed (P< 0.05) after vitrification. In conclusion, Piau blastocysts can be cryopreserved by Cryotop. This study also demonstrated that the apoptotic pathway may be responsible for the low efficiency of porcine embryo cryopreservation.(AU)

Blastocistos de suínos foram submetidos ao MALDI-TOF para se identificarem os principais fosfolipídios (PL). Depois, parte destes embriões (D6) foram vitrificados (n=52), ou permaneceram frescos (grupo controle, n=42). Após o aquecimento, os blastocistos foram cultivados in vitro para se avaliar a reexpansão e a eclosão (BE) às 24 e 48 horas. Finalmente, às 48 horas, os BE foram submetidos ao RT-qPCR em busca dos genes BCL2A1, BAK, BAX e CASP3. No MALDI-TOF, a intensidade do íon foi expressa em unidades arbitrárias. O desenvolvimento embrionário foi comparado por qui-quadrado (P<0,05). Entre os PL mais representativos estavam as fosfatidilcolinas [PC (32: 0) + H] +; [PC (34: 1) + H] + e [PC (36: 4) + H] +. Além do PL, o MALDI revelou alguns triglicerídeos (TG), incluindo PPL (50: 2) + Na +, PPO (50: 1) + Na +, PLO (52: 3) + Na + e POO (52: 2) + Na. A reexpansão não diferiu (P>0,05) entre blastocistos frescos ou vitrificados às 24 (33,3%, 32,7%) e 48 horas (2,4%, 13,5%). As taxas de eclosão foram maiores (P<0,05) para o grupo fresco comparado ao vitrificado às 24 (66,7% x 15,4%) e 48 horas (97,6% x 36,0%). O BAX estava mais expresso (P<0,05) após a vitrificação. Concluindo, os blastocistos Piau podem ser criopreservados por Cryotop. Este estudo também demonstrou que a via apoptótica pode ser responsável pela baixa eficiência da criopreservação de embriões suínos.(AU)

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The aim of this study to measure the fractions of the total serum proteins of the Campeiro horse and identify the influences of biological variants. Blood samples were taken in 138 horses of the breed Campeiro for measuring the concentration of total serum protein by the biuret method. Serum concentrations of protein fractions were measured by electrophoresis using agarose gel. Groups were formed according to age, sex and reproductive condition. The average values of serum fractions: albumin (2.85±0.36g/dl), alpha 1 (0.28±0.11g/dl), alpha 2 (0.26±0.08g/dL) beta 1 (0.57±0.15g/dl), beta 2 (0.89±0.28g/dL), gamaglobulinas (1.86±0.34g/dL), albumin/globulin ratio (0.75±0.18) and 2.5% percentile and 97.5% had slight differences in relation to the reference interval proposed for the species. They observed higher values of alpha 1 and 2 globulins in the group from that had six to eight years old and gammaglobulins in group above 13 years old. Serum protein concentrations were similar in horses and mares and between non-pregnant and pregnant. Sex and pregnancy status did not affect serum proteinogram. Alpha and gammaglobulins have higher values as the age increases. Serum proteinogram of Campeiro horses shows variations that have to be considered in the interpretation of laboratory tests.(AU)

Este trabalho tem por objetivo mensurar as frações das proteínas totais séricas de equinos Campeiros e identificar as influências de variantes biológicas. Foram colhidas amostras de sangue de 138 equinos, machos e fêmeas da raça Campeiro. A determinação da concentração de proteínas totais séricas foi realizada pelo método de biureto. As concentrações séricas das frações proteicas foram determinadas por eletroforese, utilizando-se gel de agarose. Formaram-se grupos em relação à idade, ao sexo e à condição reprodutiva. Os valores médios das frações séricas albumina (2,85±0,36g/dL), alfa 1 (0,28±0,11g/dL), alfa 2 (0,26±0,08g/dL), beta 1 (0,57±0,15g/dL), beta 2 (0,89±0,28g/dL), gamaglobulinas (1,86±0,34g/dL), relação albumina/globulina (0,75±0,18) e os percentis 2,5% e 97,5% apresentaram diferenças pontuais em relação aos intervalos propostos para a espécie. Observaram-se maiores valores de alfa 1, alfa 2 globulinas, no grupo de seis a oito anos, e de gamaglobulinas, no grupo acima de 13 anos de idade. O proteinograma sérico foi similar entre machos e fêmeas e entre fêmeas vazias e gestantes. Sexo e estado gestacional não afetaram o proteinograma sérico. Alfa e gamaglobulinas têm incrementos em função de idades crescentes. O proteinograma sérico de equinos Campeiros tem variações que devem ser consideradas em exames laboratoriais.(AU)

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RESUMO

The aim of this study to measure the fractions of the total serum proteins of the Campeiro horse and identify the influences of biological variants. Blood samples were taken in 138 horses of the breed Campeiro for measuring the concentration of total serum protein by the biuret method. Serum concentrations of protein fractions were measured by electrophoresis using agarose gel. Groups were formed according to age, sex and reproductive condition. The average values of serum fractions: albumin (2.85±0.36g/dl), alpha 1 (0.28±0.11g/dl), alpha 2 (0.26±0.08g/dL) beta 1 (0.57±0.15g/dl), beta 2 (0.89±0.28g/dL), gamaglobulinas (1.86±0.34g/dL), albumin/globulin ratio (0.75±0.18) and 2.5% percentile and 97.5% had slight differences in relation to the reference interval proposed for the species. They observed higher values of alpha 1 and 2 globulins in the group from that had six to eight years old and gammaglobulins in group above 13 years old. Serum protein concentrations were similar in horses and mares and between non-pregnant and pregnant. Sex and pregnancy status did not affect serum proteinogram. Alpha and gammaglobulins have higher values as the age increases. Serum proteinogram of Campeiro horses shows variations that have to be considered in the interpretation of laboratory tests.(AU)

Este trabalho tem por objetivo mensurar as frações das proteínas totais séricas de equinos Campeiros e identificar as influências de variantes biológicas. Foram colhidas amostras de sangue de 138 equinos, machos e fêmeas da raça Campeiro. A determinação da concentração de proteínas totais séricas foi realizada pelo método de biureto. As concentrações séricas das frações proteicas foram determinadas por eletroforese, utilizando-se gel de agarose. Formaram-se grupos em relação à idade, ao sexo e à condição reprodutiva. Os valores médios das frações séricas albumina (2,85±0,36g/dL), alfa 1 (0,28±0,11g/dL), alfa 2 (0,26±0,08g/dL), beta 1 (0,57±0,15g/dL), beta 2 (0,89±0,28g/dL), gamaglobulinas (1,86±0,34g/dL), relação albumina/globulina (0,75±0,18) e os percentis 2,5% e 97,5% apresentaram diferenças pontuais em relação aos intervalos propostos para a espécie. Observaram-se maiores valores de alfa 1, alfa 2 globulinas, no grupo de seis a oito anos, e de gamaglobulinas, no grupo acima de 13 anos de idade. O proteinograma sérico foi similar entre machos e fêmeas e entre fêmeas vazias e gestantes. Sexo e estado gestacional não afetaram o proteinograma sérico. Alfa e gamaglobulinas têm incrementos em função de idades crescentes. O proteinograma sérico de equinos Campeiros tem variações que devem ser consideradas em exames laboratoriais.(AU)

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Três experimentos foram realizados para adaptar um protocolo de sincronização de estro e da ovulação para ser utilizado em programas de inseminação artificial em tempo fixo (IATF) em vacas taurinas tropicalmente adaptadas. No Exp. 1 (crossover), vacas pluríparas Curraleiro Pé-Duro (n= 12) receberam um dispositivo intravaginal contendo 1g de P4 por oito dias e 2mg de BE intramuscular (IM) no momento da inserção do dispositivo (dia 0). No dia da remoção do dispositivo (dia 8), as fêmeas receberam 150µg de D-cloprostenol sódico e 300UI de gonadotrofina coriônica equina (eCG) IM, sendo, então, divididas aleatoriamente para receber 1mg de BE no dia 8 (BE8) ou 1mg de BE no dia 9 (BE9). A aplicação de BE no D9 atrasou a ovulação em aproximadamente 15 horas (P<0,05). No Experimento 2, foram avaliados protocolos com oito (P4D8) e nove dias (P4D9) de exposição à progesterona, resultando em parâmetros de desenvolvimento folicular e luteal semelhantes entre os tratamentos (P>0,05). No Experimento 3, os protocolos hormonais de IATF BE8 e P4D9 foram testados para a taxa de prenhez, alcançando 23% (10/43) e 20% (9/45), respectivamente (P>0,05). Embora o grupo P4D9 tenha mostrado avanço na proporção de animais que responderam ao protocolo quando comparado ao protocolo BE8, este não se refletiu em melhora na taxa de prenhez.(AU)

Three experiments were performed to adapt a synchronization protocol of estrus synchronization and ovulation to be used in fixed time artificial insemination programs (FTAI) in tropically adapted Bos taurus cows. In Exp. 1 (crossover) multiparous Curraleiro Pé-Duro cows (n= 12) received an intravaginal device containing 1g of P4 for 8 days and 2mg of EB at the time of device insertion (Day 0). On the P4 device removal (Day 8) females received 150g of D-cloprostenol Sodic and 300IU of equine chorionic gonadotropin (eCG). Then, they were randomly divided to receive 1mg of EB on Day 8 (EB8) or on Day 9 (EB9). EB9 delayed ovulation approximately 15 hours (P<0.05). In Exp. 2, protocols using progesterone for eight (P4D8) or nine days (P4D9) were evaluated, resulting in similar parameters of folicular and luteal development (P>0.05). In Exp. 3, EB8 and P4D9 protocols were used to evaluate the pregnancy rate, reaching 23% (10/43) and 20% (9/45), respectively (P>0.05). Although P4D9 protocol has shown improvement in proportion of animals that responded to the protocol when compared to EB8 protocol, it was not able to improve pregnancy rate.(AU)

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Três experimentos foram realizados para adaptar um protocolo de sincronização de estro e da ovulação para ser utilizado em programas de inseminação artificial em tempo fixo (IATF) em vacas taurinas tropicalmente adaptadas. No Exp. 1 (crossover), vacas pluríparas Curraleiro Pé-Duro (n= 12) receberam um dispositivo intravaginal contendo 1g de P4 por oito dias e 2mg de BE intramuscular (IM) no momento da inserção do dispositivo (dia 0). No dia da remoção do dispositivo (dia 8), as fêmeas receberam 150µg de D-cloprostenol sódico e 300UI de gonadotrofina coriônica equina (eCG) IM, sendo, então, divididas aleatoriamente para receber 1mg de BE no dia 8 (BE8) ou 1mg de BE no dia 9 (BE9). A aplicação de BE no D9 atrasou a ovulação em aproximadamente 15 horas (P<0,05). No Experimento 2, foram avaliados protocolos com oito (P4D8) e nove dias (P4D9) de exposição à progesterona, resultando em parâmetros de desenvolvimento folicular e luteal semelhantes entre os tratamentos (P>0,05). No Experimento 3, os protocolos hormonais de IATF BE8 e P4D9 foram testados para a taxa de prenhez, alcançando 23% (10/43) e 20% (9/45), respectivamente (P>0,05). Embora o grupo P4D9 tenha mostrado avanço na proporção de animais que responderam ao protocolo quando comparado ao protocolo BE8, este não se refletiu em melhora na taxa de prenhez.(AU)

Three experiments were performed to adapt a synchronization protocol of estrus synchronization and ovulation to be used in fixed time artificial insemination programs (FTAI) in tropically adapted Bos taurus cows. In Exp. 1 (crossover) multiparous Curraleiro Pé-Duro cows (n= 12) received an intravaginal device containing 1g of P4 for 8 days and 2mg of EB at the time of device insertion (Day 0). On the P4 device removal (Day 8) females received 150g of D-cloprostenol Sodic and 300IU of equine chorionic gonadotropin (eCG). Then, they were randomly divided to receive 1mg of EB on Day 8 (EB8) or on Day 9 (EB9). EB9 delayed ovulation approximately 15 hours (P<0.05). In Exp. 2, protocols using progesterone for eight (P4D8) or nine days (P4D9) were evaluated, resulting in similar parameters of folicular and luteal development (P>0.05). In Exp. 3, EB8 and P4D9 protocols were used to evaluate the pregnancy rate, reaching 23% (10/43) and 20% (9/45), respectively (P>0.05). Although P4D9 protocol has shown improvement in proportion of animals that responded to the protocol when compared to EB8 protocol, it was not able to improve pregnancy rate.(AU)

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The present study was aimed at evaluating the seminal plasma proteins and sperm parameters of Curraleiro Pé-Duro bulls. Semen was collected from 10 bulls by electroejaculation, and sperm parameters were evaluated in fresh and frozen-thawed semen. Seminal plasma proteins were analyzed by 2-D SDS-PAGE and mass spectrophotometry. Tools in computational biology were used to generate bioinformatic knowledge and evaluate gene ontology, protein-protein interactions, phylogenetic trees and multiple sequence alignments. Sperm motility in fresh and frozen-thawed semen was 78.8±1.8% and 21.2±1.6%, respectively. Pearson's correlations were evaluated (p<0.05). Sperm motility and vigor in fresh semen were correlated with clusterin, TIMP2 and cathepsin S (r=0.64-0.71) and sperm defects were related to inhibitor of carbonic anhydrase and BSP 5 (r=0.78-0.80). Clusterin, BSP 5, alpha-enolase, creatine kinase M-type, glyceraldehyde-3-phosphate dehydrogenase, BSP 3, albumin, and 5'-nucleotidase and legumain were correlated with acrosome intact live sperm (r=0.80-0.64). Associations were detected between sperm vigor and spermadhesin 1 (r=-0.89), and between sperm defects in fresh semen and spermadhesin 1 and clusterin (r=-0.81). Sperm motility in frozen-thawed semen was associated with BSP 1, spermadhesin 1, clusterin and spermadhesin Z13 (r=0.64-0.85). The percent of motile sperm after freeze-thawing was negatively correlated (r=-0.64) with the amount of spermadhesin 1 in the seminal plasma. Based on in silico analysis, TIMP2 interacted with BSP1, BSP3, BSP5 and metalloproteinases. Molecular functions of proteins associated with sperm parameters were binding, catalytic activity and enzymatic regulation. Amino acid sequences of spermadhesin 1 and BSP 1 from Bos taurus, and other domestic species were similar. Phylogenetic tree analysis demonstrated that clusterin from Bos taurus was related to Ovis aries and domains of clusterin, spermadhesin 1, BSP 1 and inhibitor of carbonic anhydrase were conserved as well. In summary, specific seminal proteins are associated with sperm parameters of locally-adapted bulls. Use of the endangered mammalian as a model may assist in understanding aspects of evolutionary adaptations and could improve assisted reproductive biotechnologies.

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The ability to predict the effects of climatic factors on animals and their adaptability is important for livestock production. The aim of the present study was to analyze whether existing indices are suitable for evaluating heat stress in Santa Ines and Morada Nova sheep, which are locally adapted hair sheep breeds from northeastern Brazil, and if the limits used to classify thermal stress are suitable for these breeds. Therefore, climatic, physiological, and physical parameters, as well as thermographic images, were collected in 26 sheep, 1 1/2 years old, from two genetic groups (Santa Ines 12 males and 4 females; Morada Nov. 7 males and 3 females) for 3 days in both morning (4:00 a.m.) and afternoon (2:00 p.m.) with six repetitions, totalizing 156 repetitions. Statistical analysis included correlations and broken-line regressions. Iberia and Benezra indices were the tolerance tests that best correlated with the assessed parameters. High correlations between environmental indices and rectal or skin surface temperatures was observed, which indicates that these indices can be used for Santa Ines and Morada Nova sheep raised in central Brazil. However, some indicative values of thermal discomfort are different from the existing classification. Therefore, in order to classify appropriately, the model used needs to be carefully studied, because these classifying values can vary according to the species and model. Further research is necessary to establish indicators of thermal stress for sheep breeds raised in the region.

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