R. A. Satrapa

Influence of sire breed (Bos indicus versus Bos taurus) and interval from slaughter to oocyte aspiration on heat stress tolerance of in vitro-produced bovine embryos

Based on in vitro experiments, Bos indicus embryos were more resistant to heat stress (HS) than Bos taurus embryos. To increase knowledge regarding differences between Bos indicus and Bos taurus in resistance to HS, the primary objective of this study was to determine if tolerance to HS is due to the breed, origin of the oocyte, sperm, or both. Additionally, the influence of the interval between ovary acquisition (in the abattoir) and oocyte aspiration in the laboratory, on early embryo development was ascertained. Oocytes were collected from Nelore and Holstein cows in an abattoir; 4.0 or 6.5 h later, oocytes were aspired in the laboratory, and then matured and fertilized using semen from Nelore (N), Gir (GIR), or Holstein (H) bulls. Ninety-six h post insemination (hpi), embryos with ≥ 16 cells were divided in two groups: control and HS. In the control group, embryos were cultured at 39°C, whereas in the HS group, embryos were subjected to 41°C for 12 h, and then returned to 39°C. Rates of cleavage, and formation of morula and blastocysts were higher (P < 0.05) for oocytes aspirated at 4.0 versus 6.5 h after ovaries were acquired. Heat stress decreased rates of blastocyst formation for all breeds (N × N; H × H; and H × GIR) and in both time intervals (4.0 and 6.5 h). However, N × N had higher cleavage rate (P < 0.05) in both time intervals when compared with H × H and H × GIR. In addition, Nelore oocytes fertilized with Nelore semen (N × N) had higher blastocyst yields (P < 0.05) in the control and HS group, when compared with the other two breeds (H × H and H × GIR). We concluded that the breed of origin of the oocyte was more important than that of the sperm for development of thermotolerance, because bull breed did not influence embryo development after HS, and in vitro early embryonic development was impaired by increasing (from 4 to 6.5 h) the interval between ovary acquisition and oocyte aspiration.

Effects of FGF10 on Bovine Oocyte Meiosis Progression, Apoptosis, Embryo Development and Relative Abundance of Developmentally Important Genes In Vitro

Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion-related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22 h in TCM-199 supplemented with 0, 2.5, 10 or 50 ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5 ng/ml FGF10 increased (p < 0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50 ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real-time RT-PCR showed a tendency of 50 ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10 ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.

Quadro citológico vaginal, concentração plasmática de progesterona durante a gestação e medidas fetais em paca (Cuniculus paca Linnaeus, 1766)

The objective of this work was to describe in 27 pacas (Cuniculus paca Linnaeus, 1766) at 30, 60 and 90 days (D) of pregnancy diagnosed by ultrasonography (U.S.) the cell types of the vaginal epithelium by vaginal smears, to report the vulva opening condition and the characteristics of vaginal mucus, to determine the progesterone (P4) serum concentration by radioimmunoassay, and also measure by ultrasound (U.S.) the fetuses biparietal diameter (BPD) at 60 and 90 days of pregnancy. At D30, 40% of the smears exhibited surface cells (with estrogenic characteristics) and of naked nuclei. At D60 and D90, parabasal, intermediate, superficial, and navicular cells were present in the same proportions, but endocervical cells were described in only 73.9% and 69% of those days smears, respectively. At D30 the highest proportion of navicular and surface cells differed (p < 0.05) compared with other cell types. The vaginal mucus was crystalline and fluid in 100% and 70% of females at D30 and D60, respectively. It was observed the vaginal vestibule open in around 50% of females in every day of exams. Minimum detectable P4 serum concentration was obtained on 72% and 83% of females, while the averages of the BPD were 1.25 cm (± 0.16) and 2.34 cm (± 0.25) on D60 and D90, respectively. Vaginal cytology aspects at D30, D60 and D90 and BPD provide elements to contribute to pregnancy diagnosis in paca. P4 concentration shows the need of further endocrinology of pregnancy studies of paca.

Gene expression profile in heat-shocked Holstein and Nelore oocytes and cumulus cells

The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress.

Influência da raça do touro (Bos indicus x Bos taurus) na tolerância ao estresse térmico calórico de embriões bovinos produzidos in vitro

Para melhor compreender as diferencas entre zebuinos e taurinos em relacao a resistencia ao ETC, objetivou-se verificar se a resistencia ao ETC e resultado da contribuicao genetica do oocito, do espermatozoide ou de ambos. Oocitos de vacas das racas Nelore e mesticas com fenotipo predominante da raca Holandesa preto e branco (mHPB) foram coletados, maturados e fertilizados com espermatozoide de touros das racas Nelore (N), Angus (An), Brahman (Bra) e Gir (Gir). Noventa e seis horas pos-inseminacao (hpi), embrioes > 16 celulas foram separados ao acaso em dois grupos: controle e ETC. Embrioes do grupo controle foram cultivados a 39 oC continuamente e do grupo ETC expostos a 41 oC por 12 horas, retornando a seguir para 39 oC. Nao foi observado efeito do ETC nas racas estudadas, sem reducao nas taxas de blastocisto e blastocisto eclodido. As taxas de clivagem e morula dos embrioes mHPB x Gir foram inferiores (p < 0,05) as das demais racas. As racas mHPB x N apresentaram taxas de blastocisto superiores as racas mHPB x An e mHPB x Gir (p < 0,05). Concluiu-se que a contribuicao genetica do oocito e mais importante do que a do espermatozoide, uma vez que a raca do touro nao influenciou a resistencia embrionaria ao ETC.

Sincronização do estro em pacas (Cuniculus paca L.): possíveis impactos sobre parâmetros reprodutivos e produtivos

Estrus synchronization is a reproductive biotechnology used to improve artificial insemination or pairing through the manipulation of the estrous cycle at a desirable time. Employing this technique in captive pacas (Cuniculus paca L.) is important because it creates expectation of meeting the demand for paca meat and, consequently, reduces poaching. Thus, this research aims to verify the effect of a progestogen implant associated with two doses of eCG on the synchronization and induction of fertile estrus. Twenty-seven adult pacas were used, 18 non-pregnant females and nine males, divided into three groups. G1 and G2 females groups (treatments) received 1.5 mg Norgestomet and were injected intramuscularly, seven days later, with 0.13 mg of prostaglandin. After 24 hours the implants were removed and the animals immediately received 25 IU and 50 IU of ECG intramuscularly, respectively. The mating of the three groups took place on the same days. G3 females’ group (control) showed estrus on different days after D0. Females under treatment displayed estrus only after removing the implant (D8). G1, G2, and G3 pregnancy rates were 100%, 66%, and 50%, respectively. Regarding births per parturition, 100% of G1 and G3 produced one offspring, while 50% of G2 produced two. Progestogen in the form of subcutaneous implants was effective in mimicking the luteal phase of the estrous cycle. After removal, implants favored the occurrence of a fertile estrus. As a conclusion, further studies must be conducted in order to establish in-depth possible association between 50 IU of eCG, and the occurrence of twin pregnancies.

PGFM 13,14-diidro 15-keto prostaglandin F-2 alpha release in response to oxytocin challenge during early postpartum in Nelore cows

The spermatic changes associated with the natural infection in dogs by Leishmania sp was evaluated during eight consecutive weeks, using ejaculates of six seronegative and six seropositive dogs. The samples were collected once a week and evaluated for volume, concentration, motility, vigor, sperm morphology, chromatin integrity, simultaneous evaluation of the plasmatic membrane integrity, acrosome, and mitochondrial potential. The total proteins of the seminal plasma and blood were measured. The visceral leishmaniasis caused increase of major and minor defects in spermatozoa of animals attacked by moderate to severe stages of the disease. In more advanced stages of the illness, the acrosomal and plasmatic membranes integrity was adversely affected. It was not possible to establish a pattern refering the evaluation of the mitochondrial potential. The incidence of morphological changes in the seropositive animals did not promote an increase of injuries to the chromatin. All animals with leishmaniasis presented hyperproteinemia of the semen.