Low oxygen tension during in vitro embryo production improves the yield, quality, and cryotolerance of bovine blastocysts.

Mammalian oocytes undergo maturation and fertilization in the low-oxygen (O2) environment of the oviduct. To evaluate the effect of O2 tension during in vitro maturation and fertilization on embryo yield, quality, cryotolerance, and gene expression, we matured and fertilized bovine cumulus-oocyte complexes under low (5%) or high (20%) O2 tension. Presumptive zygotes from both groups were cultured at 5% O2 for 8 days. Blastocysts were vitrified, and then warmed, and cultured for further 24 h to assess their cryotolerance. Our findings indicate that low O2 during maturation and fertilization enhances embryo development and cell count in both fresh and vitrified/warmed blastocysts. In this study, the interaction of O2 tension and status (fresh or vitrified/warmed) affected the transcript abundance of SOD2, AQP3, and BAX in blastocysts. These results highlight the role of low O2 tension during bovine maturation and fertilization and provide support to using 5% O2 throughout all stages of bovine in vitro embryo production.

Effect of creep feeding with rice bran and temporary weaning on cow-calf performance.

This study was conducted to investigate if calves supplemented with rice bran in a creep feeding (CF) system associated with temporary weaning (TW) affects the performance of the cow-calf pair grazing natural grasslands during summer. Two experiments were performed using 275 cow-calf pairs of British breeds and their crosses of different parities (primiparous [n = 188] in Experiment 1 and multiparous [n = 87] in Experiment 2). In both experiments, the factors evaluated were: (1) supplementation of calves with rice bran using CF: yes (+CF) or no (-CF); (2) TW for 14 days using nose plates: yes (+TW) or no (-TW). Four experimental groups were formed in both experiments: -CF - TW, -CF + TW, +CF - TW and +CF + TW. TW decreased the average daily weight gain and live weight of calves independently of the age of their mothers. The use of CF decreased both of these parameters in calves born to primiparous cows, and there was no response in the case of calves born to multiparous cows. A greater proportion of primiparous cows that received +CF conceived in the first 21 days of mating (+CF - TW = 63.5% and +CF + TW = 50%) than the other groups. The final pregnancy rate was similar among groups in both experiments. In conclusion, TW decreased the average daily weight gain and weaning weight of calves. Supplementation of calves born to primiparous cows with rice bran decreased their daily weight gain during the period of TW and their weaning weight, but their dams were conceived earlier.

Effect of season on germinal vesicle stage, quality, and subsequent in vitro developmental competence in bovine cumulus-oocyte complexes.

Although the reproductive performance of grazing cattle is lower in summer compared to winter, the effect of season on oocyte developmental competence has not been thoroughly examined. We measured the effect of season on oocyte chromatin compaction, cumulus cell quality, and embryonic development after in vitro fertilization. Cumulus oocytes-complexes (COCs) were collected from abattoir cows' ovaries during the winter and summer months. First, we evaluated the degree of chromatin compaction in germinal vesicle (GV) oocytes (GV1 through GV3), which is associated with different degrees of developmental competence. Then, we determined the apoptotic index in cumulus cells from immature and in vitro matured COCs. Finally, in vitro matured oocytes were fertilized to determine blastocyst rate and embryo quality. During the summer months, we observed a significantly lower proportion of oocytes reaching the GV3 stage and higher levels of DNA fragmentation in cumulus cell. As a result, blastocyst yield and quality were reduced during the summer months. In conclusion, summer negatively affected oocyte GV stage progression, cumulus cell quality, and embryo development. Increased cumulus cell DNA fragmentation during summer, may partially explain the reduced oocyte maturation capacity, considering the relevance of cumulus-oocyte communication during this stage.

Heat tolerance in cows of British breeds and their crosses with bonsmara under grazing conditions.

The objective of this study was to evaluate the influence of the increasing temperature-humidity index (THI) on the body temperature of cows from different breed groups. Vaginal temperature was monitored in cows from two breed groups: a) British breeds (Hereford, Angus and their crosses, n=4); and b) their crosses with Bonsmara (BO, n=6). Vaginal temperature in cows of British breeds was always higher (P < 0.05), and the higher temperature picks coincide with afternoon hours. British cows maintained stable their vaginal temperature if THI was lower than 72.8, but for higher values, they showed linear increases. Moreover, the threshold THI associated to increase in vaginal temperature in British breeds was 72.8. However, vaginal temperature remained unchanged in Bonsmara crosses. The present work suggests that THI greater than 72.8 could affect the body temperature in grazing cattle of British breeds but not its crosses with Bonsmara.

Effect of Ethanol on Parthenogenetic Activation and α-Tocopherol Supplementation during In Vitro Maturation on Developmental Competence of Summer-Collected Bovine Oocytes.

The use of α-tocopherol during in vitro maturation (IVM) is an alternative to minimize the adverse effects of heat stress on oocyte competence. However, α-tocopherol is diluted in ethanol, which can induce oocyte parthenogenetic activation (PA). This study aimed to evaluate the role of ethanol concentration on PA and the effect of α-tocopherol supplementation during IVM on the developmental competence and the expression of key genes in blastocysts derived from summer-collected oocytes. All in vitro embryo production was conducted at 5% O2, 5% CO2 at 38.5 °C. Experiment 1: oocytes were cultured with or without 0.05% ethanol. As positive PA control matured oocytes were subjected to 3% or 7% ethanol for 7 min. Oocytes from all groups were placed in fertilization medium (22 h) and culture medium (9 days). Ethanol at 0.05% during IVM did not induce oocyte PA, however, 3% and 7% ethanol were effective parthenogenetic inductors. Experiment 2: oocytes were cultured in maturation medium supplemented with 0, 50, 100 and 200 µM α-tocopherol, diluted in 0.05% ethanol. After in vitro fertilization and embryo culture, we assessed blastocyst apoptotic index and the transcription of a panel of genes. The results showed that supplementation with 100 µM α-tocopherol reduced apoptotic index and increased the expression of SOD2. In conclusion, 100 µM α-tocopherol, diluted in 0.05% ethanol, can be used during IVM to embryonic quality.

Time-dependent effects of heat shock on the zona pellucida ultrastructure and in vitro developmental competence of bovine oocytes.

The aim of this study was to determine the effects of different exposure lenght to heat shock (HS) during in vitro maturation (IVM) on zona pellucida (ZP) ultrastructure and developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were matured in vitro (IVM) at 38.5 °C for 24 h (control group, CG), or incubated at 41 °C (HS) for 6 h (HS-6h), 12 h (HS-12h), 18 h (HS-18h), and 22h (HS-22h) followed by incubation at 38.5 °C to complete a full 24-h period of maturation. After IVM, oocytes were subjected to scanning electron microscopy (SEM) or in vitro fertilization and culture until the blastocyst stage. For heat-shocked oocytes, with exception of those in the HS-6h group, SEM examinations revealed that ZP surfaces were rough and characterized by a presence of spongy network. Oocytes from the HS-22h group displayed an increase in the number of pores, as well as a higher proportion of oocytes with amorphous ZPs. The proportion of oocytes that reached metaphase II (MII) stage decreased in all HS groups, regardless of the duration of exposure to 41 °C. These results provide evidence that HS during IVM for 12-22 h reduces the developmental competence of bovine oocytes, increasing the percentage of oocytes with abnormal chromosomal organization, and reducing fertilization and blastocysts formation rate. The effects of HS were more pronounced for the 22-h exposure group. The damage induced by HS on oocyte function clearly increased upon exposure to elevated temperature.

The use of prostaglandins in controlling estrous cycle of the ewe: a review.

This review considers the use of prostaglandin F(2α) and its synthetic analogues (PG) for controlling the estrous cycle of the ewe. Aspects such as phase of the estrus cycle, PG analogues, PG doses, ovarian follicle development pattern, CL formation, progesterone synthesis, ovulation rate, sperm transport, embryo quality, and fertility rates after PG administration are reviewed. Furthermore, protocols for estrus synchronization and their success in timed AI programs are discussed. Based on available information, the ovine CL is refractory to PG treatment for up to 2 days after ovulation. All PG analogues are effective when an appropriate dose is given; in that regard, there is a positive association between the dose administered and the proportion of ewes detected in estrus. Follicular response after PG is dependent on the phase of the estrous cycle at treatment. Altered sperm transport and low pregnancy rates are generally reported. However, reports on alteration of the steroidogenic capacity of preovulatory follicles, ovulation rate, embryo quality, recovery rates, and prolificacy, are controversial. Although various PG-based protocols can be used for estrus synchronization, a second PG injection improves estrus response when the stage of the estrous cycle at the first injection is unknown. The estrus cycle after PG administration has a normal length. Prostaglandin-based protocols for timed AI achieved poor reproductive outcomes, but increasing the interval between PG injections might increase pregnancy rates. Attempts to improve reproductive outcomes have been directed to provide a synchronized LH surge: use of different routes of AI (cervical or intrauterine), different PG doses, and increased intervals between PG injections. Finally we present our point of view regarding future perspectives on the use of PG in programs of controlled sheep reproduction.

INSL3 in the ruminant: a powerful indicator of gender- and genetic-specific feto-maternal dialogue.

The hormone Insulin-like peptide 3 (INSL3) is a major secretory product of the Leydig cells from both fetal and adult testes. Consequently, it is a major gender-specific circulating hormone in the male fetus, where it is responsible for the first phase of testicular descent, and in the adult male. In most female mammals, circulating levels are very low, corresponding to only a small production of INSL3 by the mature ovaries. Female ruminants are exceptional in exhibiting high INSL3 gene expression by the thecal cells of antral follicles and by the corpora lutea. We have developed a specific and sensitive immunoassay to measure ruminant INSL3 and show that, corresponding to the high ovarian gene expression, non-pregnant adult female sheep and cows have up to four times the levels observed in other female mammals. Significantly, this level declines during mid-pregnancy in cows carrying a female fetus, in which INSL3 is undetectable. However, in cows carrying a male fetus, circulating maternal INSL3 becomes elevated further, presumably due to the transplacental transfer of fetal INSL3 into the maternal circulation. Within male fetal blood, INSL3 is high in mid-pregnancy (day 153) corresponding to the first transabdominal phase of testicular descent, and shows a marked dependence on paternal genetics, with pure bred or hybrid male fetuses of Bos taurus (Angus) paternal genome having 30% higher INSL3 levels than those of Bos indicus (Brahman) paternity. Thus INSL3 provides the first example of a gender-specific fetal hormone with the potential to influence both placental and maternal physiology.

Effect of a povidone-iodine intrauterine infusion on progesterone levels and endometrial steroid receptor expression in mares.

BACKGROUND: Intrauterine infusions have been widely used for the treatment of endometritis in the mare. Nevertheless, their consequences on endocrine and endometrial molecular aspects are unknown. We studied the effect of a 1% povidone-iodine solution intrauterine infusion on progesterone levels, endometrial histology and estrogen (ERα) and progesterone (PR) receptor distribution by immunohistochemistry. METHODS: Fourteen healthy mares were used in this study. Estruses were synchronized and seven mares were treated with intrauterine infusions at days 0 and 2 post ovulation of two consecutive estrous cycles. Uterine biopsy samples were taken on days 6 and 15 post ovulation. RESULTS: The treatment did not induce an inflammatory response indicating endometritis, neither affected the ERα. However, it reduced the percentage of PR positive cells (PPC) on day 6 (deep glandular epithelium, control: 95.7 vs. infused: 61.5, P < 0.05). Treated mares tended to have lower progesterone levels on day 2 (3.9 ng/ml vs. 6.6 ng/ml, P = 0.07), and higher levels on day 15 compared with controls (4.4 ng/ml vs. 1.3 ng/ml, P = 0.07). CONCLUSION: a 1% povidone-iodine infusion during days 0 and 2 post ovulation in healthy mares did not induce histological changes indicating endometritis, but altered progesterone concentrations and reduced the expression of endometrial PR at day 6 without affecting the ERα. These changes could reduce embryo survival.