Rumen-Protected Choline Improves Metabolism and Lactation Performance in Dairy Cows.

Choline is required for the synthesis of phosphatidylcholine, an important constituent of lipoproteins. Early lactation cows presumably synthesize insufficient phosphatidylcholine, and choline supplementation in a rumen-protected form might benefit metabolism and lactation. The objectives of this study were to determine the effects of feeding rumen-protected choline (RPC) on lactation and metabolism in dairy cows. In experiment 1, 369 nulliparous and parous Holstein cows housed in four pens per treatment were fed 12.9 g/day of choline as RPC from 25 days prepartum until 80 days postpartum. In experiment 2, 578 nulliparous cows housed in five pens/treatment were fed 12.9 g/day of choline as RPC in the last 22 days of gestation only. In both experiments, blood was sampled and analyzed for concentrations of nonesterified fatty acids (NEFAs) and glucose at 1, 14, and 21 days postpartum and of choline at 1 and 14 days postpartum. Blood from all cows was sampled and analyzed for concentrations of ß-OH butyrate (BHB) at 1 and 14 days postpartum. Cows with BHB > 1.2 mmol/L were classified as having hyperketonemia. Hepatic tissue was collected from 46 cows from the eight pens in experiment 1 at 9 days postpartum and analyzed for concentrations of glycogen and triacylglycerol. Milk yield and components were measured for 80 days postpartum in experiment 1, whereas only milk yield was measured in experiment 2. The pen was the experimental unit of analysis. Supplementing RPC tended to increase dry matter intake (DMI) prepartum in experiments 1 and 2 and postpartum in experiment 1. Feeding cows with RPC increased yields of 3.5% fat-corrected milk (42.8 vs. 44.8 kg/day), energy-corrected milk (38.5 vs. 40.3 kg/day), milk fat (1.52 vs. 1.61 kg/day), and true protein (1.16 vs. 1.21 kg/day) in experiment 1. Milk yield tended to be greater with RPC (26.4 vs. 27.4 kg/day) in experiment 2. Supplementing RPC increased plasma choline concentrations on day 14 postpartum in experiment 1 (3.32 ± 0.27 vs. 4.34 ± 0.28 µM) and on day 1 in experiment 2 (3.35 ± 0.16 and 13.73 ± 0.15 µM). Treatment did not affect the concentrations of glucose, NEFAs, or BHB in plasma, but the incidence of hyperketonemia was less in multiparous cows fed RPC than those fed the control in experiment 1. Feeding cows with RPC reduced hepatic triacylglycerol content and tended to reduce the ratio of triacylglycerol to glycogen and the risk of hepatic lipidosis in cows in experiment 1. The concentrations of hepatic triacylglycerol on day 9 postpartum were inversely related to those of choline in plasma on day 1 postpartum. Feeding cows with RPC improved lactation and metabolism, but more benefits were noted when it was fed before and after calving.

Lack of effect of melatonin on ovarian function and response to estrous synchronization and fixed-time AI during the nonbreeding season in lactating dairy buffalo (Bubalus bubalis).

The present study was conducted to examine whether pretreatment with melatonin would enhance ovarian follicular functions and increase response to estrous synchronization and fixed-time AI (TAI) during the nonbreeding season in lactating dairy buffalo. In Experiment 1, buffalo cows without a detectable corpus luteum (CL) were assigned on Day -20 (D-20) to three groups: control (n = 12); melatonin (n = 13); progesterone (P4) (n = 15). Cows in the melatonin group were implanted with melatonin on D-20. From D0 to D9, there was imposing of an estrous synchronization treatment regimen using either a standard Ovsynch protocol (control, melatonin) or a P4-based Ovsynch treatment regimen (P4). There were no differences (P > 0.05) among groups for the presence of a CL at D0, size of the largest follicle at D0, ovulation to GnRH injection at D0 and D9, or the time to ovulation after injection of GnRH at D9. In Experiment 2, there was imposing of the same treatment regimens as in Experiment 1, with inclusion of TAI. Females of the P4 group had a greater (P = 0.001) pregnancy/AI percentage (60 %) than those in the control (17 %) and melatonin (23 %) groups. Females of the P4 group also had a larger (P = 0.005) CL at D20 compared with those in the control and melatonin groups. Findings indicate treatment with melatonin for 20 days did not affect ovarian functions or the response to an estrous synchronization treatment regimen and TAI during the nonbreeding season in lactating dairy buffalo.

Efficiency of CellROX deep red® and CellROX orange® fluorescent probes in identifying reactive oxygen species in sperm samples from high and low fertility bulls.

Detection of reactive oxygen species (ROS) is of great interest in semen analysis since their excess is detrimental to sperm function and male fertility. Fluorescence microscopy has achieved attention for providing broad possibilities of sperm evaluations and also for presenting substantial accessibility. In this context, this study investigated the efficiency of CellROX Deep Red® and Orange® probes in detecting ROS in bovine sperm cells and assessed their relationship with sperm fertility potential. First, 16 ejaculates were assigned in three treatments: T0 (no ROS production induced), T1x (ROS production induced once) and T2x (ROS production induced twice). Samples were incubated with Red and Orange probes and percentages of cells producing ROS were evaluated using fluorescence microscopy. Coefficient of determination was 0.61 for Red and 0.56 for Orange. Afterwards, frozen-thawed semen samples from high and low fertility bulls were evaluated regarding percentages of cells producing ROS detected by Red and Orange. Higher levels of ROS assessed by Red were detected in low fertility bovine samples. In conclusion, CellROX Red® and Orange® are both efficient in detecting ROS in bovine spermatozoa. Furthermore, higher sperm ROS detection by CellROX Red® might be associated with low fertility samples.

Sperm-borne miR-216b modulates cell proliferation during early embryo development via K-RAS.

Semen fertilizing potential is dependent upon the morphological, functional and molecular attributes of sperm. Sperm microRNAs (miRNAs) were recently shown to hold promise regarding their association with different fertility phenotypes. However, their role in fertility regulation remains to be determined. We postulated that sperm miRNAs might regulate early embryonic development. From this perspective, sperm quality and 380 sperm miRNAs were investigated in frozen-thawed semen from high (HF; 54.3 ± 1.0% pregnancy rate) and low (LF; 41.5 ± 2.3%) fertility bulls. Out of nine miRNAs that showed different levels in sperm cells, miR-216b was present at lower levels in HF sperm cells and zygotes. Among miR-216b target genes (K-RAS, BECN1 and JUN), K-RAS, related to cell proliferation, revealed a higher level in HF two-cell embryos. First cleavage rate, blastocyst cell number and division number were also higher in HF. In addition, by using a model based on polyspermy embryos, we demonstrated an increase in miR-216b levels in zygotes associated with sperm cell entry. Our results shed light on a possible mechanism of paternal contribution involving sperm-borne miR-216b that modulates levels of miR-216b in zygotes and K-RAS in two-cell embryos. This modulation might regulate early development by interfering with the first cleavage and blastocyst quality.

Resynchronization improves reproductive efficiency of suckled Bos taurus beef cows subjected to spring-summer or autumn-winter breeding season in South Brazil.

The objective of the present study was to evaluate the effect of estrus resynchronization (RE) after the first Timed Artificial Insemination (TAI) protocol on reproductive efficiency of suckled Bos taurus beef cows (n = 1052; Angus and Brangus) subjected to two different breeding season (BS) in Rio Grande do Sul, Brazil. In experiment 1, cows were maintained in native pasture on conventional BS (spring-summer), and in experiment 2, cows were maintained in a grazing area cultivated in an integrated crop-livestock system on modified BS (autumn -winter). Experiment 1 cows (n = 92) were randomly distributed in two reproductive managements: 1) TAI at the first day of the BS (Day 0), followed by natural service (NS) on day 10 until the end of the BS (TAI + NS; n = 45) or, 2) TAI on day 0, followed by RE 22 days after the first TAI and then followed by NS on day 42 until the end of the BS (TAI + RE22 + NS; n = 47). Experiment 2 cows (n = 960) were subjected to one of four reproductive managements: 1) only NS for 90 days of the BS (NS; n = 266); 2) TAI on day 0, followed by NS on day 10 until the end of the BS (TAI + NS; n = 200); 3) TAI on day 0, followed by RE 22 days after the first TAI and then followed by NS on day 42 until the end of the BS (TAI + RE22 + NS; n = 249) or, 4) TAI on day 0, followed by RE 30 days after the first TAI and then followed by NS on day 50 until the end of the BS (TAI + RE30 + NS; n = 245). In experiment 1, conception rate after first TAI was similar for treatments TAI + NS (42%, 19/45) and TAI + RE22 + NS (48%, 23/47; P = 0.4107). Total pregnancy rates at day 60 of the BS were 55.6% and 66.6% for TAI + NS and TAI + RE22 + NS, respectively (P = 0.006). In experiment 2, only 3% of the females submitted to NS were pregnant 30 days after the onset of BS, a lower rate compared to other groups (TAI + NS = 40%; TAI + RE22 + NS = 39.8%; TAI + RE30 + NS = 40%; P < 0.001). At 70 days of the BS, the groups TAI + RE22 + NS and TAI + RE30 + NS obtained greater pregnancy rate (66.3% and 69.4%, respectively) than other groups (NS = 16.9%; TAI + NS = 48%; P < 0.001). Pregnancy rate at the end of BS was greater in RE groups (TAI + RE22 + NS = 81.5%; TAI + RE30 + NS = 83.7%) than the NS (45.1%) or TAI + NS (71%). In conclusion, resynchronization increases pregnancy rate per AI (P/AI) and reproductive efficiency of suckled Bos taurus beef cows during spring-summer or autumn-winter breeding season in natural pasture or cultivated pasture, respectively.

Impact of hormonal modulation at proestrus on ovarian responses and uterine gene expression of suckled anestrous beef cows.

BACKGROUND: This study evaluated the impact of hormonal modulation at the onset of proestrus on ovarian response and uterine gene expression of beef cows. METHODS: A total of 172 anestrous beef cows were assigned to one of four groups according to the treatment with estradiol cypionate (ECP) and/or equine chorionic gonadotropin (eCG) [CON (n = 43), ECP (n = 43), eCG (n = 44) and ECP + eCG (n = 42)]. RESULTS: ECP-treated cows (ECP and ECP + eCG groups) presented greater occurrence of estrus (44.6% vs. 65.4%; P = 0.01) and pregnancy per AI [47.1% vs. 33.3%; P = 0.07], but similar progesterone (P4) concentration at subsequent diestrus than cows not treated with ECP (CON and eCG groups). Nonetheless, eCG-treated cows (eCG and ECP + eCG groups) presented larger follicle at timed AI (12.6 ± 0.3 vs. 13.5 ± 0.3 mm; P = 0.03), greater ovulation rate (96.5% vs. 82.6%; P = 0.008) and greater P4 concentration at d 6 (3.9 ± 0.2 vs. 4.8 ± 0.2 ng/mL; P = 0.001) than cows not treated with eCG (CON and ECP groups). Next, cows with a new corpus luteum 6 d after TAI were submitted to uterine biopsy procedure. Uterine fragments [CON (n = 6), ECP (n = 6)] were analyzed by RNA-Seq and a total of 135 transcripts were differentially expressed between groups (73 genes up-regulated by ECP treatment). Subsequently, uterine samples were analyzed by qPCR (genes associated with cell proliferation). ECP treatment induced greater abundance of PTCH2 (P = 0.07) and COL4A1 (P = 0.02), whereas suppressed EGFR (P = 0.09) expression. Conversely, eCG treatment increased abundance of HB-EGF (P = 0.06), ESR2 (P = 0.09), and ITGB3 (P = 0.05), whereas it reduced transcription of ESR1 (P = 0.05). Collectively, supplementation with ECP or eCG at the onset of proestrous of anestrous beef cows influenced ovarian responses, global and specific endometrial gene expression. CONCLUSION: Proestrus estradiol regulate the endometrial transcriptome, particularly stimulating proliferative activity in the endometrium.

Impact of spontaneous Neospora caninum infection on pregnancy loss and subsequent pregnancy in grazing lactating dairy cows.

The impact of spontaneous Neospora caninum infection on pregnancy loss and subsequent pregnancy in grazing lactating dairy cows was evaluated. Data from 1273 females (878 multiparous and 395 first-calving cows) from six preselected dairy herds were analyzed. Cows were classified as seropositive (SP) (prevalence, 24%; range, 11%-33%) or seronegative (SN) by indirect immunofluorescence detection of antibodies against N caninum. Seropositive cows (prevalence, 40.0%) presented higher (P < 0.001) incidence of abortion compared with SN cows (prevalence, 4.1%). Neospora caninum DNA was detected by real-time polymerase chain reaction in 44.4% of intact aborted fetuses from SP cows, whereas none was found in those aborted from SN cows. The average daily milk production adjusted to 305 days was lower (P < 0.001) in SP (22.5 ± 0.3 L/day) than in SN cows (24.8 ± 0.2 L/day). Furthermore, SP cows presented greater occurrence of retained placenta (17.1% vs. 6.0%; P < 0.001) and acute postpartum metritis (9.8% vs. 2.4%; P < 0.001). Despite similar pregnancy rates after first postpartum artificial insemination (27.6% vs. 31.8%; P = 0.40), cumulative pregnancy rates during 300 days in milk (94.7% vs. 98.5%; P = 0.005) were greater in SN cows. A reduced (P = 0.0001) Cox proportional hazard of pregnancy rate at 300 days in milk and a longer interval from parturition or abortion to conception (median, 111 vs. 101 days) were observed in SP compared with SN cows. Spontaneous N caninum infection is a significant contributing factor of pregnancy loss and occurrence of uterine disease (i.e., retained placenta and metritis), negatively affecting subsequent pregnancy in grazing lactating dairy cows.

Ovarian responses of dairy buffalo cows to timed artificial insemination protocol, using new or used progesterone devices, during the breeding season (autumn-winter).

This study evaluated the effect of new or used P4 devices on the ovarian responses of dairy buffalo that were administered an estradiol (E2) plus progesterone (P4)-based timed artificial insemination (TAI) protocol during the breeding season. On the first day of the TAI protocol, 142 cows were randomly assigned to receive one of the following: a new device (New; 1.0 g of P4; n = 48); a device that had previously been used for 9 days (Used1x, n = 47); or a device that had previously been used for 18 days (Used2x, n = 47). Ultrasound was used to evaluate the following: the presence of a corpus luteum (CL); the diameter of the dominant follicle (ØDF) during protocol; ovulatory response; and pregnancies per AI (P/AI). Despite similar responses among the treatments, there was a significant positive association of the ØDF during TAI protocol with ovulatory responses and number of pregnancies. In conclusion, satisfactory ovarian responses and a satisfactory pregnancy rate were achieved when grazing dairy buffalo were subjected to the TAI protocol in breeding season, independent of whether a new or used P4 device was used. Furthermore, the presence of the larger follicle was associated with a higher ovulation rate and higher P/AI following TAI.

Manifestation of estrous behavior and subsequent progesterone concentration at timed-embryo transfer in cattle are positively associated with pregnancy success of recipients.

Plasma estradiol and progesterone (P4) concentrations during the peri-ovulatory period are positively correlated with pregnancy success in cattle. The aims of this study were to assess the effects of estrus occurrence and early diestrus P4 concentrations on pregnancy per timed-embryo transfer (P/TET). A total of 267 crossbred beef heifers [222 with corpus luteum (CL) and 45 without a CL but with a follicle >8mm at beginning of the estrous synchronization protocol) received an intra-vaginal P4 device and intramuscular administration of estradiol benzoate. Progesterone devices were removed 8 days later (Day 0), and heifers received d-cloprostenol, eCG and estradiol cypionate. Estrous behavior was monitored twice daily for 3 days after P4 device removal. Plasma P4 concentration was measured by radioimmunoassay at Day 7 and Day 9. At Day 9, heifers with a CL (n=236; i.e. submission rate of 85.5%; 236/276) undergoing TET received an in vitro-produced embryo. Heifers expressing a standing behavioral estrus had a greater P/TET than heifers that did not express a standing estrus [62.4% (106/170) compared with 47.0% (31/66)]. The probability of pregnancy was positively correlated with plasma P4 concentration at TET. When heifers were grouped by quartiles of P4 concentration at TET (Q1=0.64±0.16, Q2=1.70±0.04, Q3=2.90±0.07 and Q4=5.52±0.27ng/mL) the P/TET were 45.8% (Q1; 27/59)(c), 52.25% (Q2; 31/59)(bc), 66.1% (Q3; 39/59)(ab) and 67.8% (Q4; 40/59)(a). Additionally, heifers that became pregnant had greater P4 concentrations at TET (2.87±0.16ng/mL; n=137) than heifers that did not become pregnant (2.45±0.24ng/mL; n=99). No statistical difference was observed regarding P4 concentrations on Day 7, regardless of standing estrus or pregnancy status. In cattle, manifestation of estrous behavior and plasma P4 concentration at TET increase the probability of pregnancy in in vitro-produced embryo recipients.

In vitro oocyte transport through the oviduct of buffalo and crossbred beef cows / Transporte de oócitos in vitro através do oviduto de búfalas e vacas de corte cruzadas

The present study was conducted to verify if the elevation of plasma concentrations of estradiol during superovulatory treatments affects the oocyte transport in buffalo females, as well as if the inferior quality of buffalo oocytes and/or some functional difference on the oviduct of these animals is responsible for the low embryo recovery rate in superovulated buffaloes when compared to cows subjected to the same treatment. Oviducts of 10 buffaloes and 15 of cows, treated to induce a single ovulation were used. The oviducts were placed on Petri dishes and received the following treatments: 5 buffalo oocytes with no E2 (G-BufBuf and G-BovBuf), 5 bovine oocytes with no E2 (G-BufBov and G-BovBov), 5 buffalo oocytes with E2 (G-BufE2Buf and G-BovE2Buf) and 5 bovine oocytes with E2 (G-BufE2Bov and G-BovE2Bov; factorial 2x2x2). Oocytes were incubated for 24h. Subsequently, oviducts were washed and oocytes were recovered and counted. Since no interactions were found between E2 treatment, oviducts and oocytes species, main effects were analyzed separately. Recovery rate and number of oocytes was higher on cattle compared to buffaloes (35.0+8.6% and 1.4+0.3 vs. 10.0±4.6% and 0.5±0.2, respectively; p<0.05); no effect of E2 treatment was observed on recovery rate and number of oocytes (29.8±9.0% and 1.3±0.4 vs. 16.9±6.1% and 0.7±0.2, respectively; p>0.05); the number of buffaloes and bovine oocytes recovered were similar (1.4±0.4 and 0.6±0.2, respectively; p>0.05). Oocytes recovery rate showed a trend (P=0.07) to be higher when buffalo oocytes were implanted when compared to bovine oocytes (35.2±9.2% vs. 12.9±5.4%). Present results suggest that oocyte transport by the oviduct of buffaloes and bovine was not dependent on oocytes species or E2 supplementation to the culture medium.

O presente estudo foi realizado para verificar se a elevação das concentrações plasmáticas de estradiol durante os tratamentos superovulatórios afeta o transporte dos oócitos em fêmeas bubalinas, bem como se a qualidade inferior dos oócitos de búfalos e/ou alguma diferença funcional no oviduto destes animais é responsável pela baixa taxa de recuperação de embriões em búfalas superovuladas quando comparadas a vacas submetidas ao mesmo tratamento. Foram utilizados 10 ovidutos de búfalas e 15 de vacas, tratadas para a indução de ovulação única. Os ovidutos foram colocados em placas de Petri e receberam os seguintes tratamentos: sem E2 e inseridos com 5 oócitos de búfalas (G-BufBuf e G-BovBuf); sem E2 e com 5 oócitos de vacas (G-BufBov e G-BovBov); com E2 e com 5 oócitos de búfalas (G-BufE2Buf e G-BovE2Buf); e com E2 e com 5 oócitos de vacas (G-BufE2Bov e G-BovE2Bov; fatorial 2x2x2). Posteriormente, foram incubados por 24h e, após esse período, foram lavados para a recuperação e contagem dos oócitos. Como não foi verificado efeito de interação, foram analisados os efeitos principais. O número e a taxa de recuperação de oócitos foi maior em ovidutos de vacas que de búfalas (1,4±0,3/35,0±8,6% vs. 0,5±0,2/10,0±4,6%; P<0,05). Foi verificado que o tratamento com ou sem E2 não interferiu no número e na taxa de recuperação de oócitos (1,3±0,4/29,8±9,0% vs. 0,7±0,2/16,9±6,1%; P>0,05). Não foi verificada diferença no número de oócitos de búfalas ou de vacas recuperados (1,4±0,4 e 0,6±0,2; P>0,05). Observou-se também que houve tendência (P=0,07) de maior taxa de recuperação de oócitos de búfalas que de vacas (35,2±9,2% vs. 12,9±5,4%). Os dados são indicativos de que o transporte de oócitos pelo oviduto de búfalas e de vacas independe da espécie do oócito e não é influenciado pelo E2.

Xenooplasmic transfer between buffalo and bovine enables development of homoplasmic offspring.

Nuclear-mitochondrial incompatibilities may be responsible for the development failure reported in embryos and fetuses produced by interspecies somatic cell nuclear transfer (iSCNT). Herein we performed xenooplasmic transfer (XOT) by introducing 10 to 15% of buffalo ooplasm into bovine zygotes to assess its effect on the persistence of buffalo mitochondrial DNA (mtDNA). Blastocyst rates were not compromised by XOT in comparison to both in vitro fertilized embryos and embryos produced by transfer of bovine ooplasm into bovine zygotes. Moreover, offspring were born after transfer of XOT embryos to recipient cows. Buffalo mtDNA introduced in zygotes was still present at the blastocyst stage (8.3 vs. 9.3%, p = 0.11), indicating unaltered heteroplasmy during early development. Nonetheless, no vestige of buffalo mtDNA was found in offspring, indicating a drift to homoplasmy during later stages of development. In conclusion, we show that the buffalo mtDNA introduced by XOT into a bovine zygote do not compromise embryo development. On the other hand, buffalo mtDNA was not inherited by offspring indicating a possible failure in the process of interspecies mtDNA replication.

Perfil de FSH e LH na divergência folicular em novilhas Nelore (Bos indicus) / FSH and LH profiles during follicle deviation in Nelore heifers (Bos indicus)

O presente estudo teve como objetivo traçar o perfil de gonadotrofinas em 12 novilhas Nelore, a fim de testar a hipótese de que há declínio nas concentrações de FSH e elevação transitória nos níveis de LH durante a seleção folicular. A partir da ovulação (D0) foram colhidas amostras de sangue da veia jugular a cada 12 horas até o D5, para a dosagem de FSH e LH plasmáticos. Empregou-se o método de radioimunoensaio de duplo anticorpo. A sensibilidade do ensaio de LH foi de 0,02ng/ml e a de FSH de 0,005ng/ml. Os coeficientes de variação intra-ensaio foram 13,6% e 18,8%, respectivamente. Os dados (média±EPM) foram normalizados para o momento da divergência folicular e, posteriormente, analisados por ANOVA e por regressão inear, cúbica e quadrática. Também foi empregado o Teste t para comparação entre o ponto mais alto e o mais baixo da curva. Não se verificou efeito de tempo sobre as concentrações plasmáticas de FSH e de LH quando se utilizou análise de variância e de regressão. Entretanto, através do Teste t pontual, o FSH atingiu as menores concentrações plasmáticas 36 (0,40±0,05ng/ml) e 60 horas (0,42±0,04ng/ml) após a divergência, comparativamente às 36 horas anteriores ao desvio, quando as concentrações foram máximas (0,63±0,08ng/ml). Conclui-se, portanto, que há declínio nas concentrações plasmáticas de FSH, contudo, não foi comprovada elevação transitória nas concentrações de LH próximo ao momento do desvio folicular em fêmeas Nelore.

Present study aimed to evaluate gonadotropins profiles in 12 Nelore heifers, in order to test the hypothesis that FSH concentrations decrease and LH presents a transient increase during follicle selection. Blood samples from jugular vein were harvested twice daily starting at the time of ovulation (D0) until D5. Plasma samples were assayed for FSH and LH by double antibody radioimmunoassay method. LH and FSH assay sensitivity was 0,02ng/ml and 0,005ng/ml, respectively. The intraassay coefficient of variation was 13,6% and 18,8%, respectively. Data (mean±SEM) were normalized to follicle deviation and analyzed by ANOVA and by linear, cubic, and quadratic regressions. Comparisons between higher and lower FSH values were also performed by T-test. There was no effect of time in plasmatic FSH and LH circulating levels when variance analysis or regression analysis were performed. However, by T-test, FSH concentrations reached the lowest plasmatic levels 36 (0,40±0,05ng/ml) and 60 hours (0,42±0,04ng/ml) after follicular deviation, comparatively to 36 hours before deviation, when the concentrations were maximal (0,63±0,08ng/ml). In conclusion, there is a FSH decrease, although a transient LH elevation has not been confirmed encompassing follicle deviation in Nelore females.

Produção in vitro de embriões bovinos com soro de égua ou de vaca em estro com ou sem a adição de LH/FSH / In vitro production of bovine embryos with oestrus mare's serum or oestrus cow serum with or without LH/FSH

Mil duzentos e setenta e um oócitos foram divididos em 4 tratamentos com a finalidade de se avaliar a influência da adição de LH e FSH na produção in vitro de embriões bovinos com soro de vaca em estro (SVE) ou soro de éguas obtido no 1º dia do estro (SE). Independente do tratamento os oócitos foram maturados com TCM199 + 5,95mg/ml de Hepes, 0,025mg/ml de piruvato de sódio e 2,2mg/ml de bicarbonato de sódio, sendo adicionado 10 por cento de soro de égua (ES), 10 por cento de SVE (VS), 10 por cento de soro de égua + 0,5mg/ml de hormônio luteinizante bovino (LHb) + 0,01UI/ml de hormônio folículo estimulante recombinante humano-rFSHh (EH) e 10 por cento SVE + LHb + rFSHh (VH). Os oócitos assim tratados, foram maturados em estufa com 5 por cento de CO2 a 39ºC sob umidade saturada por 22-24h. Depois, foram fecundados em TALP-FERT por 18-20h e cultivados por 8 dias em meio SOF + 5 por cento de SE (ES e EH) ou SVE (VS e VH). As taxas de clivagem de 72 por cento obtidas no grupo VH (229/316) e 61 por cento no grupo VS (193/315) foram significativamente menores (p<0,05) que as dos grupos ES (80 por cento - 254/317) e EH (80 por cento - 257/323). A produção de embriões (blastocistos iniciais, blastocistos, blastocistos expandidos e eclodidos) no D7 após a inseminação para os grupos ES (32 por cento), EH (28 por cento) e VH (27 por cento), foi significativamente superior aos 20 por cento obtidos no VS (p<0,05). No D9, verificou-se uma diferença significativa (p<0,05) entre os grupos ES (31 por cento) e EH (29 por cento) quando comparados com o grupo VS (22 por cento), mas não houve diferença quando comparamos com o VH (24 por cento). A taxa de eclosão em D9 foi de 11 por cento (ES), 11 por cento (EH), 10 por cento (VS) e 5 por cento (VH). Não houve diferença entre as médias do número de células dos embriões (Blastocistos expandidos e eclodidos) obtidos no D9. Conclui-se, com estes resultados que, para a obtenção de taxas regulares de blastocistos, não seja necessária a adição de hormônios quando se utilize o soro de égua, e que os hormônios devam ser adicionados quando se utilize soro de vaca.

Produção in vitro de embriões bovinos em tubos sem controle da atmosfera gasosa / In vitro production of bovine embryo in tubes without gaseous atmosphere control

Com a maior utilização da OPU existe a necessidade de encontrar alternativas para iniciar as primeiras fases da PIV sem controle da atmosfera. O desenvolvimento de um método prático e simples de transporte/maturação/fecundação permitiria maior eficiência do laboratório e diminuição dos custos de produção. O objetivo deste estudo foi desenvolver um método de maturação e fecundação para complexos cumulus oócitos (CCO) em tubos de polipropileno sem gaseificação, mantidos em banho-maria. A maturação in vitro em estufa foi conduzida em TCM-199 modificado (controle) enquanto que para os tratamentos em banho-maria, em tubos, o meio foi acrescido de 25mM de N-2-hidroxietilpiperazina-N'-2-ácido etanosulfônico (HEPES). Na fecundação in vitro dos oócitos em tubos, os CCO foram mantidos em banho-maria a 39ºC em Talp-Fert, acrescido de HEPES em concentrações entre 10mM a 28,7 mM por 10 a 18 horas, sob óleo mineral. O cultivo realizou-se em placas em bolsas gaseificadas com meio SOFaaci com soro de vaca em estro (SVE), sob óleo mineral, em estufa com 5,00 por centoCO2 , 5,00 por centoO2 e 90,00 por cento de N2 a 39ºC, por 9 dias. Verificou-se que CCO maturados por 24h em tubos não gaseificados, mantidos em banho-maria também podem ser fecundados em meio Talp-Fert com 25 mM de HEPES, em tubos não gaseificados, mantidos em banho-maria durante 10 horas. As taxas de clivagem, blastocistos em D7 e D9 em tubos não gaseificados foram semelhantes (P>0,05) aos procedimentos em estufa. A fecundação em Talp-Fert com 10 mM a 28,7 mM de HEPES em tubos mantidos em banho-maria prejudica o desenvolvimento embrionário quando conduzida por 18 horas.