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.

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.

Thermoregulation of male sheep of indigenous or exotic breeds in a tropical environment.

Climate change has intensified the frequency of heat waves in the world, thereby exposing farm animals to stressful conditions. For better productive performance it is important to identify the most resilient genotypes. Thus, our objective was to evaluate the thermoregulatory responses of rams of tropical indigenous (Morada Nova and Santa Inês) and exotic breeds (Dorper and Texel), by monitoring the environmental and physiological indicators related to heat tolerance. The experiment was carried out in a tropical climate region (Cwa), in Brazil, for twelve months, which comprised spring, summer, autumn and winter. Thirty-three rams were divided into groups: Morada Nova (MN; n=8, red-coat), Santa Inês (SI; n=9, black-coat), Dorper (DO; n=8, white-coat) and Texel (TX; n=8, white-coat). The microclimatic variables were monitored, and the THI and BGHI comfort indices were calculated. Coat thickness and body surface temperatures were measured monthly, and serum triiodothyronine-T3 measurements and complete blood tests were performed. The physiological variables were evaluated every fifteen days and skin micro-biopsies were performed in the summer and winter for histological evaluation. During the warmer seasons, the THI and BGHI reached values that indicated thermal discomfort. TX showed higher coat thickness throughout the year, increased physiological variables related to thermolysis, and reduced T3 (P < 0.05). The internal temperature was permanently lower in the MN, SI and DO (P < 0.05). The body surface temperatures were affected by the coat characteristics and wool length. Hematological parameters varied in the seasons with the highest thermal conditions (P < 0.05). The MN showed larger sweat glands, while the area occupied by the sweat glands was higher in the SI. The DO showed higher hair density in the summer and winter (P < 0.05). The results indicated that the MN, SI and DO breeds overcome the thermal challenge more easily throughout the seasons due to specific adaptive morphological and physiological characteristics.

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.