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.

Puberty Blocker, Leuprolide, Reduces Sex Differences in Rough-and-Tumble Play and Anxiety-like Behavior in Juvenile Rats.

We examined the effect of the puberty blocker, leuprolide acetate, on sex differences in juvenile rough-and-tumble play behavior and anxiety-like behavior in adolescent male and female rats. We also evaluated leuprolide treatment on gonadal and pituitary hormone levels and activity-regulated cytoskeleton-protein messenger RNA levels within the adolescent amygdala, a region important both for rough-and-tumble play and anxiety-like behavior. Our findings suggest that leuprolide treatment lowered anxiety-like behavior during adolescent development, suggesting that the maturation of gonadotropin-releasing hormone systems may be linked to increased anxiety. These data provide a potential new model to understand the emergence of increased anxiety triggered around puberty. Leuprolide also reduced masculinized levels of rough-and-tumble play behavior, lowered follicle-stimulating hormone, and produced a consistent pattern of reducing or halting sex differences of hormone levels, including testosterone, growth hormone, thyrotropin, and corticosterone levels. Therefore, leuprolide treatment not only pauses sexual development of peripheral tissues, but also reduces sex differences in hormones, brain, and behavior, allowing for better harmonization of these systems following gender-affirming hormone treatment. These data contribute to the intended use of puberty blockers in stopping sex differences from developing further with the potential benefit of lowering anxiety-like behavior.

Efeito da tosquia na temperatura corpórea e outros parâmetros clínicos em ovinos / Effect of shearing on the skin temperature and other clinical parameters in sheep

Os objetivos deste estudo foram determinar a variação da temperatura após a tosquia de ovinos em condição ambiental quente e seca e comparar as mudanças de temperatura com variações de frequências cardíaca e respiratória, movimentos ruminais e estado de hidratação. Vinte ovelhas Suffolk não tosquiadas foram estudadas. O exame físico foi realizado em todos os animais três vezes ao dia às 7:00, 1:00 PM e 7:00 PM, durante 42 dias (22 dias antes do corte e 20 dias após o corte). A temperatura da superfície corporal foi medida pelo termómetro de infravermelho sobre vários pontos. Os dados foram submetidos à análise de variância, para comparações entre os grupos (tosquiado x não tosquiado) em cada tempo, e a diferença significativa foi avaliada em nível de P<0,05 pelo teste de Tukey. A frequência respiratória foi estatisticamente significativa em todos os horários de cólera (P<0,05). Quando a umidade do ar estava alta, as freqüências respiratórias foram baixas (P= -1). O estresse térmico foi claro em ovinos deste estudo, refletindo mudanças acentuadas nas frequências cardíaca e respiratória e temperatura retal. A frequência respiratória foi o parâmetro mais confiável para estabelecer um quadro de estresse térmico em ovelhas não tosquiadas, com valores em média de três vezes maior do que os relatados na literatura. A correlação entre as temperaturas de superfície corpórea com a temperatura ambiente e umidade do ar foi negativa, explicado pelo efeito de isolamento de lã, ou seja, mesmo com um aumento da temperatura ambiente e umidade, a temperatura do corpo tende a manter um equilíbrio de compensação. Nos animais tosquiados, a correlação entre a temperatura da pele com a temperatura ambiente e umidade do ar mostraram que a temperatura da pele aumenta quando aumenta a temperatura ambiente. O aumento da temperatura ambiente não afeta a temperatura do corpo dos animais não tosquiados devido ao efeito isolante da lã. No entanto, quando a temperatura ambiente sobe, a presença da lã começa a afetar o conforto térmico, já que a presença desta dificulta a perda de calor pela sudação. Neste estudo, os melhores indicadores de estresse térmico foram a frequência respiratória e temperaturas retais e da pele. As temperaturas da pele medidas na face interna da coxa, axilas e períneo foram consideradas as mais confiáveis.

The aim of this study was to determine the variation of the temperature after shearing in sheep under dry and hot environment conditions and to compare the temperature changes with variation in cardiac and respiratory frequencies, ruminal movements and hydration status. Twenty Suffolk unshorn ewes were studied. Physical examination was performed in all animals three times a day at 7:00 AM, 1:00 PM and 7:00 PM, during 42 days (22 days before shearing and 20 days after shearing). The skin temperature was measured by infrared thermometer over several surfaces of the body. Data were submitted to analysis of variance, for comparisons between groups (shorn versus unshorn) at each time, and the significant difference was evaluated at level of P<0.05 by Tukey test. The respiratory frequency was statistically significant at all times. When air humidity was high, the respiratory frequencies were low. The thermal stress was clear in sheep of this study, reflecting marked changes in cardiac and respiratory frequencies and rectal temperature. The respiratory frequency was the parameter more reliable to establish a framework of thermal stress in the unshorn sheep, with values on average three times higher than those reported in the literature. The heart rate monitors the thermal variation of the environment and is also an indicator of heat stress. This variation shows the Suffolk breed is well adapted to hot climates. The correlation between the body surface temperatures with environment temperature and air humidity was negative, as explained by the effect of wool insulation, i.e. even with an increase in environment temperature and humidity, the body temperature tends to maintain a compensating balance. In the shorn animals, the correlation between skin temperature with environment temperature and air humidity showed that the skin temperature increases when the environment temperature increases. The increase in the environment temperature does not affect the body temperature of unshorn animals due the insulating effect of the wool. However, when environment temperature rises, the presence of the wool starts to affect the thermal comfort as the heat absorption is larger than the capacity of heat loss. In this study, the best thermal stress indicators were the respiratory frequency and rectal and skin temperatures. The temperatures of the skin measured at the perineum, axillae and inner thigh were considered the most reliable.