Unidade de Pesquisa e Desenvolvimento de Registro

Registro, Brazil

Unidade de Pesquisa e Desenvolvimento de Registro

Registro, Brazil

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Macabelli C.H.,Federal University of São Carlos | Macabelli C.H.,University of Sao Paulo | Ferreira R.M.,University of Sao Paulo | Gimenes L.U.,São Paulo State University | And 12 more authors.
PLoS ONE | Year: 2014

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis. © 2014 Macabelli et al.


Carvalho N.A.T.,Unidade de Pesquisa e Desenvolvimento de Registro | Soares J.G.,University of Sao Paulo | Souza D.C.,Escritorio de Desenvolvimento Rural de Registro | Vannucci F.S.,University of Sao Paulo | And 5 more authors.
Theriogenology | Year: 2014

Three experiments were designed to evaluate the effect of different circulating progesterone (P4) concentrations during synchronization of ovulation protocol for timed artificial insemination of seasonal anestrous buffalo cows. In the first trial, ovariectomized cows were randomly allocated into one of three groups: using new P4 devices (G-New; n = 8), using devices previously used for 9 days (G-Used1x; n = 8), and using devices previously used for 18 days (G-Used2x; n = 8). The P4 device was maintained for 9 days, and the circulating P4 concentration was measured daily. The circulating P4 concentrations during the P4 device treatment were the lowest for G-Used2x (1.10 ± 0.04 ng/mL), intermediate for G-Used1x (1.52 ± 0.05 ng/mL), and the highest for G-New (2.47 ± 0.07 ng/mL; P = 0.001). In the second trial, 31 anestrous cows had their ovarian follicular dynamics evaluated after receiving the treatments described previously (G-New [n = 10], G-Used1x [n=11], and G-Used2x [n = 10]). At insertion of the P4 device, cows were administered 2.0 mg of estradiol benzoate. Nine days later, the P4 device was removed and cows were administered 0.53 mg of cloprostenol sodium plus 400 IU of eCG. Forty-eight hours after P4 device removal, 10 μg of buserelin acetate was administered. There were no differences among the groups (G-New vs. G-Used1x vs. G-Used2x) in diameter of the largest follicle at P4 device removal (9.0 ± 0.8 vs. 10.1 ± 0.9 vs. 8.6 ± 0.8 mm; P = 0.35), in interval from P4 device removal to ovulation (77.1 ± 4.5 vs. 76.5 ± 4.7 vs. 74.0 ± 4.4 hours; P = 0.31), or in ovulation rate (80.0% vs. 81.8% vs. 60.0%; P = 0.51). In experiment 3, 350 anestrous cows were randomly assigned into one of the three treatments described previously (G-New,n= 111; G-Used1x, n = 121; G-Used2x, n = 118) and received a timed artificial insemination for 16 hours after buserelin treatment. The 30-day pregnancy rates did not differ among groups (55.9% vs. 55.4% vs. 48.3%; P = 0.39). Thus, the low circulating P4 concentrations released from a used P4 device efficiently control the ovarian follicular growth and had no detrimental effect on the pregnancy rates of the seasonal anestrous buffalo cows. © 2014 Elsevier Inc.


Carvalho N.A.T.,Unidade de Pesquisa e Desenvolvimento de Registro | Gimenes L.U.,University of Sao Paulo | Soares J.G.,State University of Maranhão | Souza D.C.,University of Sao Paulo | And 3 more authors.
Revista Veterinaria | Year: 2010

We hypothesized that dominant follicle diameter, ovulation and pregnancy rate would be increased to a greater extent when GnRH was administered at 56h after PGF2á, 8h after the usual administration at the Ovsynch protocol. To verify our hypothesis, 128 buffalo were assigned into two Groups (G48 and G56) and received 10mg of GnRH on Day 0 (D0). On D7, a PGF2a doses was administered. Animals of G48 and G56 received more 10mg of GnRH at 48h and 56h after the PGF2á, respectively (D9). In both groups the FTAI was accomplished 16h after the last GnRH administration. Third seven buffaloes were assigned to ultrasonography examination (Mindray DP2200Vet, 7,5MHz) on D7 and from D9 to D12. The pregnancy diagnoses were performed by ultrasound on D40. Data were analysed by SAS for Windows program. There were no significant differences between G48 and G56 on dominant follicle diameter (14.2 ± 0.4 vs. 14.2 ± 0.9 mm), ovulation (76.5 ± 1.1 vs. 70.0 ± 1.1 %) and pregnancy rate (50.8 vs. 50.7 %; P>0.05). However, the delay of 8h on GnRH administration caused an anticipation of ovulation on G56 compared to G48 (24.0 ± 3.8 vs. 33.7 ± 2.6 h; P<0.05). In conclusion, we found no advantage to delay the last GnRH administration at the Ovsynch protocol in buffalo, not confirming our hypothesis. Moreover, an inconvenience of the Ovsynch-56 compared to Ovsynch-48 is handling buffaloes in both the morning and evening during reproductive management protocols.


de Carvalho N.A.T.,Unidade de Pesquisa e Desenvolvimento de Registro | Soares J.G.,University of Sao Paulo | de Souza D.C.,EDR CATI | Maio J.R.G.,Ourofino Agronegocio | And 4 more authors.
Buffalo Bulletin | Year: 2013

The present study aimed to evaluate the effect of using the intravaginal progesterone device for eight or nine days on the follicular response and pregnancy rate in lactating buffaloes synchronized for timed artificial insemination (TAI) during non breeding season. Two hundred and twenty buffaloes were randomly assigned according to age, parity, days postpartum, body condition score and ovarian activity into two groups: intravaginal progesterone device for 8 days (G-8d, n=110) or for 9 days (G-9d, n=110). At random stage of the estrous cycle (D0, PM) the buffaloes (G-9d) received an intravaginal progesterone device (P4) used a third time and 2.0 mg i.m. of estradiol benzoate (EB). On day 1 PM, buffaloes from G-8d received the same treatment described above for G-9d. On D9 (PM), buffaloes received 0.53 mg i.m. of PGF2 (Cloprostenol sodic) and 400 IU of eCG, followed by P4 device removal. After 48 h (D11, PM), the ovulation was induced by the administration of 10 μg i.m. of GnRH (buserilin acetate). In a subset of the buffaloes (G-8d, n=12 and G-9d, n=12), transrectal ovarian ultrasound examinations were performed on D0 to verify ovarian activity, on D9 to check the diameter of largest follicle and from D11 to D14 (12/12h for 60h) to verify the disappearance of the ovulatory follicle. All animals were submitted to TAI 64 h after P4 removal (D12, AM). Pregnancy examinations were conducted by transrectal ultrasonography 30 days after TAI. The variables were analyzed using the GLIMMIX procedure of SAS®. There were no difference between experimental groups (G-8d and G-9d) for all variables analyzed (P>0.05): diameter () of the largest follicle in D9 (9.3±0.7 vs. 10.3±0.4mm); of the ovulatory follicle (15.4±0.7 vs. 14.4±0.4mm); interval between P4 removal and ovulation (73.5±3,1 vs. 76.8±3.8h); ovulation rate [66.7% (8/12) vs. 83.3% (10/12)]; pregnancy rate [42.7% (47/110) vs. 50.9% (56/110)]. It was concluded that third used intravaginal P4 devices for eight or nine days resulted in satisfactory follicular response and pregnancy rate of the synchronized lactating buffaloes for TAI during the non breeding season. However, the 8.2% decreasing pregnancy rate in the G-8d should be considered in future investigations.


Carvalho N.A.T.,Unidade de Pesquisa e Desenvolvimento de Registro | Gimenes L.U.,University of Sao Paulo | Reis E.L.,University of Sao Paulo | Cavalcante A.K.S.,University of Sao Paulo | And 10 more authors.
Revista Veterinaria | Year: 2010

In order to analyse anatomic particularities between buffalo and bovine females, the genital systems from 31 cyclic buffalo and 21 cyclic bovine were morphometrycally evaluated. The genital systems were dissected and the organs were separated and measured. The ovaries weight, the right ovary length and wideness, the number of cervix rings, the vagina and cervix length were higher in bovine than buffalo. Otherwise, the ovaries thickness, the left oviduct length, the uterine body, the left horn length and wideness were higher in buffalo than bovine. The left ovary length and wideness, the right oviduct length, the right uterine horn length and wideness did not differ between species. The knowledge of the anatomic particularities is important to the employment of reproductive techniques like artificial insemination and embryo transfer on these species.


Carvalho N.A.T.,Unidade de Pesquisa e Desenvolvimento de Registro | Gimenes L.U.,University of Sao Paulo | Ayres H.,University of Sao Paulo | Soares J.G.,State University of Maranhão | And 3 more authors.
Revista Veterinaria | Year: 2010

The aim of this study was to evaluate the effect of eCG treatment on follicular response, diameter of CL and P4 level on buffaloes synchronized with P4 device. Forty buffalo were assigned into two Groups (GnoeCG and GeCG) and received a P4 device (DIB®) plus 2.0mg of estradiol benzoate (D0). On D9, the DIB® was removed and a PGF2a was administered. On this day, buffalo in GeCG received 400IU of eCG. After two days (D11), each buffalo received 10μg of GnRH. Buffaloes were examined by ultrasonography on D -12, D0, D9, D11 to D14, D16, D20 and D24. All animals were bled in order to measure P4 level from D16 to D24. There were no significant differences between GnoeCG and GeCG on dominant follicle diameter, device removal/ovulation interval, ovulation rate, CL diameter and volume on D20 and D24. Notwithstanding, there were differences between GnoeCG and GeCG on CL diameter and volume on D16 and P4 level from D16 to D24. These results indicate that treatment with eCG can increase the CL diameter and volume and the P4 level on anestrous buffaloes synchronized with P4 device. The beneficial effect of eCG needs to be further investigated on pregnancy rate of anestrous buffaloes submitted to FTAI.


Baruselli P.S.,University of Sao Paulo | Soares J.G.,University of Sao Paulo | Gimenes L.U.,São Paulo State University | Monteiro B.M.,University of Sao Paulo | And 2 more authors.
Buffalo Bulletin | Year: 2013

Currently, timed ovulation induction and timed artificial insemination (TAI) can be performed in buffalo using GnRH or estradiol plus progesterone/progestin (P4)-releasing devices and prostaglandin F2α (PGF2α). The control of the emergence of follicular waves and of ovulation at predetermined times, without the need for estrus detection, has facilitated the management and improved the efficiency of AI programs in buffalo during the breeding and nonbreeding season. Multiple ovulations, embryo transfer, ovum collection and in vitro embryo production have been shown to be feasible in buffalo, although low efficiency and limited commercial application of these techniques have been documented as well. These results could be associated with low ovarian follicular pools, high levels of follicular atresia and failures of the oocyte to enter the oviduct after superstimulation of follicular growth. This review discusses a number of key points related to the manipulation of ovarian follicular growth to improve pregnancy rates following TAI and embryo transfer of in vivo- and in vitro-derived embryos in buffalo.

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