Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries.

BACKGROUND AND AIMS: Ovum pick-up (OPU) is an intrinsic step of in vitro fertilization procedures. Nevertheless, it can cause ovarian lesions and compromise female fertility in bovines. Recently, we have shown that intraovarian injection of adipose-derived mesenchymal stromal cells (AD-MSCs) effectively preserves ovarian function in bovines. Given that MSC-derived extracellular vesicles (MSC-EVs) have been shown to recapitulate several therapeutic effects attributed to AD-MSCs and that they present logistic and regulatory advantages compared to AD-MSCs, we tested whether MSC-EVs would also be useful to treat OPU-induced lesions. METHODS: MSC-EVs were isolated from the secretome of bovine AD-MSCs, using ultrafiltration (UF) and ultracentrifugation methods. The MSC-EVs were characterized according to concentration and mean particle size, morphology, protein concentration and EV markers, miRNA, mRNA, long noncoding RNA profile, total RNA yield and potential for induction of the proliferation and migration of bovine ovarian stromal cells. We then investigated whether intraovarian injection of MSC-EVs obtained by UF would reduce the negative effects of acute OPU-induced ovarian lesions in bovines. To do so, 20 animals were divided into 4 experimental groups (n = 5), submitted to 4 OPU cycles and different experimental treatments including vehicle only (G1), MSC-EVs produced by 7.5 × 106 AD-MSCs (G2), MSC-EVs produced by 2.5 × 106 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 106 AD-MSCs, injected after OPU sessions 1, 2 and 3 (G4). RESULTS: Characterization of the MSC-EVs revealed that the size of the particles was similar in the different isolation methods; however, the UF method generated a greater MSC-EV yield. MSC-EVs processed by both methods demonstrated a similar ability to promote cell migration and proliferation in ovarian stromal cells. Considering the higher yield and lower complexity of the UF method, UF-MSC-EVs were used in the in vivo experiment. We evaluated three therapeutic regimens for cows subjected to OPU, noting that the group treated with three MSC-EV injections (G4) maintained oocyte production and increased in vitro embryo production, compared to G1, which presented compromised embryo production following the OPU-induced lesions. CONCLUSIONS: MSC-EVs have beneficial effects both on the migration and proliferation of ovarian stromal cells and on the fertility of bovines with follicular puncture injury in vivo.

Mecanismos epigenéticos nos espermatozoides e o impacto sobre a fertilidade masculina / Epigenetic mechanisms on sperm cells and the impact on male fertility

Por ser uma célula altamente especializada, o espermatozoide apresenta diferentes mecanismos epigenéticos, sendo os principais as metilações do DNA, o código de histonas, os ncRNAs (RNAs não codificadores), e a alta condensação da cromatina pela presença das protaminas. Estes mecanismos interagem entre si, contribuindo para a formação do epigenoma espermático, que modela a carga molecular espermática, que, por sua vez, pode impactar sobre as características do desenvolvimento embrionário e da progênie. Dessa forma, atualmente é consenso que o papel do espermatozoide ultrapassa a entrega de DNA de qualidade para o oócito no momento da fecundação. Pesquisas recentes de diversos grupos, incluindo o nosso, mostram que além da contribuição com DNA de qualidade, o espermatozoide entrega moléculas ao oócito no momento da fecundação que influenciam o desenvolvimento do embrião. Recentemente, essas moléculas de origem espermática (Em inglês: sperm-borne) também são associadas com alterações metabólicas e cognitivas da progênie. Embora ainda pouco se entenda como esses mecanismos podem persistir mesmo com o ciclo de reprogramação celular que ocorre logo após a fecundação, é evidente que estes podem impactar as características da progênie. Nesta revisão abordaremos sobre a modulação do epigenoma espermático e seus efeitos no desenvolvimento embrionário.(AU)

Since it is a highly specialized cell, the spermatozoa display different epigenetic mechanisms; the main ones are DNA methylation, histone code, ncRNAs (non-coding RNAs), and high chromatin condensation by the presence of protamines. These mechanisms act in synergy contributing to forming the sperm epigenome, which modulates the spermatic molecular cargo, and, may impact embryo and offspring development features. Thus, it is currently a consensus that the role of spermatozoa goes beyond delivering quality DNA to the oocyte at fertilization. Relevant findings from several research groups, including ours, have shown that sperm delivers several molecules to the oocyte at fertilization, beyond the contribution to DNA, which influences the development of the embryo. Recently, these sperm-borne molecules have also been associated with metabolic and cognitive changes in the offspring. Although the mechanism by which these changes can persist even after embryo reprogramming is not completely understood, evidence shows that sperm cell molecular content impacts embryo and offspring development. This review will mainly focus on the modulation of the sperm epigenome and its effects on embryo development.(AU)

Estresse calórico e reprodução em ovinos: fundamentos e perspectivas tecnológicas / Heat stress on sheep reproduction: fundamentals and technological perspectives

A ovinocultura é uma importante atividade que produz proteínas de alto valor biológico, mas seus ganhos podem ser reduzidos em função do estresse ambiental. Isso reforça a importância de se estudar a relação entre o ambiente térmico e o animal, identificando animais mais adaptados e férteis, e melhores práticas de manejo. O ovino (Ovis aries) é um animal homeotérmico, que mantem sua temperatura corporal em equilíbrio dinâmico. Quando em estresse calórico, os ovinos usam mecanismos sensíveis e latentes para dissipar o calor acumulado, com destaque para o redirecionamento do fluxo sanguíneo, a ofegação e a sudorese. O escroto desempenha importante crucial na termorregulação dos testículos, os quais precisam funcionar sob em até 6,0oC abaixo da temperatura interna corpórea. A hipertermia testicular compromete a espermatogênese, reduz a concentração seminal, a motilidade progressiva e a viabilidade espermática. Ainda, leva a aumento dos defeitos morfológicos espermáticos, na produção de espécies reativas de oxigênio e na fragmentação do DNA espermático, diminuindo a capacidade fecundante. Tecnologias disruptivas para monitoramento do ambiente de produção, da termorregulação e do bem-estar dos animais já são realidade e se encontram em expansão, favorecendo a tomada de decisões em tempo real e o desempenho reprodutivo dos ovinos.

Sheep farming is a relevant activity that provides proteins of high biological value, but its gains can be reduced due to environmental stress. This reinforces the importance of studying the relationship between the thermal environment and the animal, identifying more adapted and fertile animals, and better management practices. Sheep (Ovis aries) are homeothermic animals and thus maintain their body temperature in a state of dynamic balance. Under heat stress, sheep dissipate accumulated heat through sensitive and latent mechanisms, primarily using redirection of blood flow, panting and sweating. The scrotum plays a crucial role in the thermoregulation of the testicles, which need to be maintained up to 6.0oC below the body core temperature. Testicular hyperthermia impairs spermatogenesis, reduces seminal concentration, progressive motility and sperm viability. Furthermore, it leads to an increase in sperm morphological defects, in the production of reactive oxygen species, and in sperm DNA fragmentation, reducing the fertilizing capacity. Disruptive technologies for monitoring the production systems, animal thermoregulation and welfare are already a reality and are expanding, favoring realtime decision making and the reproductive performance of sheep.

Differences in the thermal sensitivity and seminal quality of distinct ovine genotypes raised in tropical conditions.

For different ovine breeds to maximize their reproductive capacity in countries with tropical climate, it is important to evaluate their potential for thermal resilience and consequences on their reproductive traits. Therefore, the objective of this study was to evaluate the effect of thermal environment temperatures of climate seasons in a tropical climate region on the surface temperatures of the scrotum, testicular biometric characteristics, seminal quality and serum testosterone concentration of rams of different genotypes. Breeders of four different genotypes (Dorper, n = 8, Texel, n = 8, Santa Inês, n = 9 and Morada Nova, n = 8) were used throughout the four climate seasons. Higher thermal challenge was recorded in the spring and summer. In the summer increase in scrotal surface temperature was detected by infrared thermography (P < 0.05), mainly in the regions of the distal testicular pole and tail of the epididymis. The animals of the Texel genotype had higher rectal temperature in the summer. In spring, this genotype also had the highest testicular pole (32.2 ±â€¯0.5 °C; P < 0.05) and distal (29.9 ±â€¯0.4 °C; P < 0.05) temperatures and a higher mean testicular temperature (31.7 ±â€¯0.4 °C; P < 0.05). The Morada Nova genotype showed a higher surface temperature gradient between testicular poles (2.96 ±â€¯0.1 °C; P < 0.05), especially in spring. Genotype-dependent thermal sensitivity was detected for the thermal gradient between the testicular poles, reflecting the seminal quality. There was a positive correlation of the thermal gradient between testicular poles with sperm membrane integrity and negative correlation with total sperm defects. The Texel genotype showed less progressive motility and higher percentage of sperm defects. There was no difference in testosterone concentration between genotypes and in the different seasons (P > 0.05). Thus, the indigenous genotypes showed a greater capability to maintain the scrotum-testicular thermoregulation. Dorper animals resembled the indigenous sheep genotypes, in terms of seminal characteristics, unlike Texel animals, which showed lower adaptability and lower seminal quality.

Thermoregulatory responses and reproductive traits in composite beef bulls raised in a tropical climate.

It is believed that increased livestock production is limited by tropical climate. Thermal imbalance in bulls can lead to hyperthermia and alter testicular metabolism, causing subfertility or infertility. Therefore, the thermoregulation of composite Canchim bulls (5/8 Charolais × 3/8 Zebu) raised in tropical climate as well as their consequences in the physiological, hematological, hormonal, and andrological parameters were evaluated monthly. The bulls (n = 18; 30.0 ± 1.5 months; 503.8 ± 23.0 kg) were kept on pasture, in a single group, from August 2015 to March 2016, comprising the winter, spring, and summer seasons. Biometeorological variables were continuously monitored, and the Temperature and Humidity Index (THI) was calculated. A greater thermal challenge occurred in spring and summer (THI ≥ 72.0). Nevertheless, the bulls exhibited normothermia (38.6 to 38.9 °C) in these seasons. The cortisol did not vary between seasons (7.0 vs. 8.7 vs. 6.8 ng/mL; P > 0.05) and remained within the physiological patterns. Independent of the seasons, stress leukogram was also not observed, refuting the incidence of acute or chronic thermal stress. It is noteworthy that T3 and testosterone increased (P < 0.0001, P < 0.05) in spring and summer, the time that coincides with the breeding season, when there is increased metabolic requirement from the bulls. The progressive thermal challenge increase did not affect the scrotal thermoregulatory capacity, and in general, scrotal temperature remained at 5.2 °C below the internal body temperature. In summer, there was a 5% reduction in the minor sperm defects (P < 0.05) and DNA fragmentation in 2.4% of spermatozoa, a compatible value for high fertility bulls. The results show that the studied composite bulls can be considered as climatically adapted and constitute a viable alternative to be used in production systems in a tropical climate, even if the breeding seasons occur during the most critical thermal condition periods of the year.

Use of two doses of cloprostenol in different intervals for estrus synchronization in hair sheep under tropical conditions.

This study evaluated the effect of two doses of prostaglandin at different intervals on reproductive parameters of crossbred ewes. In Experiment 1, 30 ewes received two doses of 120 µg cloprostenol at 7 (G 7 days), 9 (G 9 days), or 11.5 (G 11.5 days) days apart. Ultrasound assessments were performed from the first and second cloprostenol administration for 5 days or ovulation detection. Estrus signs were checked by a teaser male. Plasma progesterone concentration was measured before each cloprostenol dose. In Experiment 2, 95 ewes were allocated into the same treatments and after the second dose, ewes in estrus were mated. At 30 days after breeding, pregnancy diagnosis was conducted and prolificacy was evaluated at lambing. In Experiment 1, at the first cloprostenol administration, 50% of ewes had an active CL and all showed estrus. At the second administration, 66.7% of ewes had an active CL and one did not present estrus. There was no difference (P > 0.05) after the second dose for as follows: overall estrous response (90%), interval from cloprostenol administration to estrous onset (42.0 ± 4.9 h), estrus duration (31.5 ± 2.1 h), ovulation rate (100.0%), and number of ovulations (1.5 ± 0.3). In Experiment 2, both pregnancy and prolificacy rates were similar (P > 0.05) for G 7 days (73.3; 145%), G 9 days (75.9; 125%), or G 11.5 days (75.9; 145%), leading to an overall pregnancy rate of 75.0% (66/88) and prolificacy rate of 137%. Therefore, the three treatments proposed were able to promote high pregnancy and prolificacy rates in crossbred ewes.