Time filter

Source Type

Vilarino M.,Institute Reproduccion Animal Uruguay | Menchaca A.,Institute Reproduccion Animal Uruguay
Theriogenology | Year: 2013

The objective was to determine serum progesterone concentrations, ovarian responses, and pregnancy rate in sheep inseminated following a short-term protocol (6 days of treatment) with a previously used controlled internal drug release-G (CIDR-G) device. In experiment 1, 30 ewes were put on a short-term protocol using a CIDR-G of first use (new devices, N = 10), second use (previously used for 6 days, N = 10), or third use (previously used twice for 6 days, N = 10). All ewes were given prostaglandin F2α (10 mg dinoprost) and eCG (300 IU) im at device withdrawal. Mean serum progesterone concentrations were greater for ewes treated with new versus reused devices (P < 0.05), but there were no significant effects on ovarian follicular development, namely: proportion of ewes that reached ovulation/treated ewes (8/10, 9/10, and 10/10); day of emergence of the ovulatory follicle (2.9 ± 1.8, 1.8 ± 2.4, and 2.5 ± 1.1 days after CIDR-G insertion); and lifespan of the ovulatory follicle (5.4 ± 1.8, 6.5 ± 2.2, and 5.6 ± 1.0 days) for devices of first, second, and third use, respectively. The proportion of ewes that ovulated from a new follicle emerging after CIDR-G insertion was 100% (10/10) with new devices, and 77.8% (7/9) and 80% (8/10) with devices used two and three times, respectively (P = not significant). In experiment 2, ewes (N = 319) received the short-term protocol as in experiment 1, using CIDR-G of first or third use (N = 158 and N = 161, respectively) for 6 days, with prostaglandin F2α and eCG given im at device withdrawal. Intrauterine (laparoscopy) fixed-time AI with fresh semen (100 × 106 sperm) was done 52 to 57 hours after device withdrawal. Pregnancy rates were 80.4% (127/158) and 71.4% (115/161) for devices of first and third use, respectively (P = 0.06). We concluded that the CIDR-G with short-term protocol was effective for estrous synchronization and ovulation, with lower serum progesterone concentrations for reused devices. Three times used CIDR-G yielded a pregnancy rate >70%, which tended to be lower than that obtained with new devices, adding evidence of the detrimental effect of low serum progesterone concentrations on fertility in sheep. © 2013 Elsevier Inc.


Olivera-Muzante J.,Area de Produccion y Sanidad Ovina | Gil J.,Area de Teriogenologia | Fierro S.,Area de Produccion y Sanidad Ovina | Menchaca A.,Institute Reproduccion Animal Uruguay
Theriogenology | Year: 2011

The objective was to improve the reproductive performance of a prostaglandin (PG) F 2α-based protocol for timed artificial insemination (TAI) in sheep (Synchrovine®: two doses of 160 μg of delprostenate 7 d apart, with TAI 42 h after second dose). Three experiments were performed: Experiment 1) two doses of a PGF 2α analogue (delprostenate 80 or 160 μg) given 7 d apart; Experiment 2) two PGF 2α treatment intervals (7 or 8 d apart) and two times of TAI (42 or 48 h); and Experiment 3) insemination 12 h after estrus detection or TAI with concurrent GnRH. Experiments involved 1131 ewes that received cervical insemination with fresh semen during the breeding season (32/34 °S-58 °W). Estrous behaviour, conception rate, prolificacy, and fecundity (ultrasonography 30-40 d), were assessed. In Experiment 1, ewes showing estrus between 25 and 48 h or at 72 h after the second PGF 2α did not differ between 80 and 160 μg of delprostenate (73 vs 86%, P = 0.07; and 92 vs 95%, P = NS, respectively). Conception rate and fecundity were lower (P < 0.05) using 80 vs 160 μg (0.24 vs 0.42, and 0.27 vs 0.47, respectively). In Experiment 2, giving PGF 2α 7 d apart resulted in higher (P < 0.05) rates of conception (0.45 and 0.51) and fecundity (0.49 and 0.53) than treatments 8 d apart (conception: 0.33 and 0.29; fecundity: 0.33 and 0.34) for TAI at 42 and 48 h, respectively. In Experiment 3, rates of conception, prolificacy and fecundity were similar (NS) between Synchrovine® with TAI at 42 h (0.50, 1.13, and 0.56) and AI 12 h after estrus detection (0.47, 1.18, and 0.55), and Synchrovine® plus GnRH at TAI (0.38, 1.28, and 0.49). However, all TAI treatments had lower (P < 0.05) prolificacy and fecundity compared to AI following detection of spontaneous estrus (1.39 and 0.83, respectively). In conclusion, the Synchrovine® protocol was: a) more successful using 160 vs 80 μg delprostenate; b) more successful with a 7 d than 8 d PGF 2α interval; c) similarly effective for TAI versus AI 12 h after estrus detection; and d) not improved by giving GnRH at TAI. © 2011 Elsevier Inc.


Vilarino M.,Institute Reproduccion Animal Uruguay | Menchaca A.,Institute Reproduccion Animal Uruguay
Small Ruminant Research | Year: 2010

Four experiments were conducted to evaluate the effectiveness of a new controlled drug releasing device containing 0.3 g progesterone (DICO®) on ovarian control in sheep. In experiment 1, serum progesterone concentrations induced by a 14 days treatment of DICO® (n = 9) and CIDR-G® (n = 9) were compared in ovariectomized ewes. Both devices induced similar responses and no differences were recorded. In experiment 2, the onset of oestrus and the time of ovulation obtained after 14 days treatment with DICO® (n = 8) and CIDR-G® (n = 7) were compared in cyclic ewes. Both devices induced oestrus and ovulation in all of the ewes. The onset of oestrus (34.5 ± 2.8 and 30.0 ± 7.7 h), the time of ovulation (60.0 ± 9.1 and 54.9 ± 6.4 h), the ovulation rate (1.3 ± 0.5 and 1.4 ± 0.5), the follicular diameter at ovulation (7.0 ± 0.8 and 7.3 ± 1.1 mm), and the lifespan of the ovulatory follicles (8.6 ± 2.2 and 10.0 ± 2.9 days) were similar for the DICO® and CIDR-G® devices, respectively. In Experiment 3, the re-utilization of DICO® devices inserted for 6 days (i.e. short-term protocol) was evaluated in ovariectomized ewes. The females received a re-used (previously used for 6 days; n = 11) or a new DICO® (n = 11) for a period of 6 days. The re-used DICO® devices induced a lower serum progesterone concentration than the new devices (P < 0.05). However, the re-used DICO® device maintained serum progesterone concentrations above 7.1 nmol/L (i.e. >2 ng/ml) throughout treatment. In Experiment 4, the administration of eCG treatment at DICO® withdrawal was evaluated in cyclic ewes. The short-term protocol using DICO® devices for 6 days was applied with (n = 8) or without (n = 7) 300 IU eCG at the time of device withdrawal. The administration of eCG advanced ovarian follicular development, synchronizing the onset of oestrus at 36 h and the time of ovulation at 60 h from device withdrawal. In conclusion, data from these experiments show the use of DICO® or CIDR-G® devices containing 0.3 g of progesterone to have a similar efficiency in controlling serum progesterone concentrations, follicular development and the time of ovulation in sheep. The re-use of the devices, associated with the short-term protocol for 6 days is possible, although further studies on induced fertility rates are warranted. © 2010 Elsevier B.V. All rights reserved.


Vilarino M.,Institute Reproduccion Animal Uruguay | Menchaca A.,Institute Reproduccion Animal Uruguay
Theriogenology | Year: 2011

Because intravaginal devices impregnated with 0.3 g of progesterone (i.e., CIDR-G) contain remaining hormone after their use in a Short-term Protocol (5 to 7 d of treatment), the reuse of these devices is proposed in goats. Two experiments were designed to study the effects of the reutilization of CIDR-G, establishing serum progesterone concentrations, follicular development, ovulatory response, and fertility. Experiment 1: Thirty dairy goats received a Short-term Protocol for 5 d using CIDR-G of first use (new devices, n = 10), second use (previously used for 5 d, n = 10), or third use (previously used twice for 5 d each time, n = 10). Goats were given (im) prostaglandin F2α (10 mg dinoprost) and eCG (300 IU) at device insertion and withdrawal, respectively. Serum progesterone concentrations induced by CIDR-G of first use were higher than CIDR-G of second or third use (P < 0.05); concentrations were consistently > 1 ng/mL in all females treated with reused devices. Estrus and ovulation were synchronized in 100% of goats (no differences among treatments). All females treated with new devices, but only 80% of females treated with re-used devices ovulated a new follicle that emerged after CIDR-G insertion (P = NS). Ovulation occurred between 60 and 70 h after device removal (no differences among groups). Experiment 2: In goats subjected to a Short-term Protocol followed by AI at 54 h after CIDR-G, pregnancy rates with CIDR-G of first, second, and third use were 75.3% (64/85), 67.4% (60/89), and 62.1% (54/87), respectively (devices of first versus third use, P < 0.05). In summary, intravaginal devices originally containing 0.3 g of progesterone appeared effective to synchronize estrus and ovulation after first, second and third use in the Short-term Protocol. Although the pregnancy rate with reused devices was acceptable (i.e., > 60%), it was significantly lower than that achieved with new devices and further studies to ensure adequate follicular turnover are required. © 2011 Elsevier Inc.


Nunez-Olivera R.,Institute Reproduccion Animal Uruguay | De Castro T.,Institute Reproduccion Animal Uruguay | Garcia-Pintos C.,Institute Reproduccion Animal Uruguay | Bo G.,Institute Reproduccion Animal Cordoba | And 2 more authors.
Animal Reproduction Science | Year: 2014

The objective of this study was to evaluate the effect of equine chorionic gonadotropin (eCG) administration associated to fixed-time AI (FTAI) on follicular dynamics, ovulation, corpus luteum (CL) development and serum progesterone concentrations. Multiparous suckled Hereford cows (n=46) in anestrus with 60-75 days postpartum were used. Females received an intravaginal device containing 0.5g of progesterone during 8 days and 2mg of estradiol benzoate i.m. at device insertion. At device removal 500μg of cloprostenol and 0.5mg of estradiol cypionate were administered i.m., and FTAI was performed 52-56h later. Cows were divided into two experimental groups to receive 400IU of eCG i.m. at device removal (n=23), while control group did not receive eCG (n=23). Daily ovarian ultrasonography (7.5MHz transducer) and progesterone concentrations determined by RIA were assayed from device removal until 30 or 14 days after FTAI, respectively. Treatment with eCG increased ovulation rate [65.2% (15/23) vs. 30.4% (7/23); P=0.018], ovulatory follicle diameter (14.5±0.4 vs. 13.1±0.7mm, mean±SEM; P=0.081), CL area from 6 to 14 days after FTAI (344.3±25.1 vs. 274.2±23.9mm2; P=0.045) and mean serum progesterone concentrations from FTAI to 14 days later (3.0±0.2 vs. 1.8±0.2ng/ml; P=0.001), in comparison with control cows. In conclusion, the addition of eCG to a progesterone and estradiol' based treatment for FTAI improves ovulation rate and luteal function in anestrous cows. These findings have implications in order to increase pregnancy rates in FTAI treatments in Bos taurus beef cattle. © 2014 Elsevier B.V.


PubMed | Institute Reproduccion Animal Uruguay, National University of Villa María, University of Sao Paulo and National University of Cordoba
Type: Journal Article | Journal: Theriogenology | Year: 2016

Fixed-time artificial insemination (FTAI) has been regarded as the most useful method to increase the number of cows inseminated in a given herd. The main treatments for FTAI in beef cattle are based on the use of progesterone-releasing devices and GnRH or estradiol to synchronize follicle wave emergence, with a mean pregnancy per AI (P/AI) around 50%. However, more recent protocols based on GnRH (named 5-day Co-Synch) or estradiol (named J-Synch) that reduce the period of progesterone device insertion and extend the period from device removal to FTAI have been reported to improve P/AI in beef cattle. Furthermore, treatments to resynchronize ovulation for a second FTAI in nonpregnant cows have provided the opportunity to do sequential inseminations and achieve high P/AI in a breeding season, reducing or even eliminating the need for clean-up bulls. In summary, FTAI protocols have facilitated the widespread application of AI in beef cattle, primarily by eliminating the necessity of estrus detection in beef herds.


PubMed | Institute Pasteur Of Montevideo, Institute Reproduccion Animal Uruguay, Roslin Institute, French Institute of Health and Medical Research and McGill University
Type: Journal Article | Journal: Theriogenology | Year: 2016

Genetically engineered sheep and goats represent useful models applied to proof of concepts, large-scale production of novel products or processes, and improvement of animal traits, which is of interest in biomedicine, biopharma, and livestock. This disruptive biotechnology arose in the 80s by injecting DNA fragments into the pronucleus of zygote-staged embryos. Pronuclear microinjection set the transgenic concept into peoples mind but was characterized by inefficient and often frustrating results mostly because of uncontrolled and/or random integration and unpredictable transgene expression. Somatic cell nuclear transfer launched the second wave in the late 90s, solving several weaknesses of the previous technique by making feasible the transfer of a genetically modified and fully characterized cell into an enucleated oocyte, capable of cell reprogramming to generate genetically engineered animals. Important advances were also achieved during the 2000s with the arrival of new techniques like the lentivirus system, transposons, RNA interference, site-specific recombinases, and sperm-mediated transgenesis. We are now living the irruption of the third technological wave in which genome edition is possible by using endonucleases, particularly the CRISPR/Cas system. Sheep and goats were recently produced by CRISPR/Cas9, and for sure, cattle will be reported soon. We will see new genetically engineered farm animals produced by homologous recombination, multiple gene editing in one-step generation and conditional modifications, among other advancements. In the following decade, genome edition will continue expanding our technical possibilities, which will contribute to the advancement of science, the development of clinical or commercial applications, and the improvement of peoples life quality around the world.


Crispo M.,Institute Pasteur Of Montevideo | Mulet A.P.,Institute Pasteur Of Montevideo | Tesson L.,French Institute of Health and Medical Research | Barrera N.,Institute Reproduccion Animal Uruguay | And 7 more authors.
PLoS ONE | Year: 2015

While CRISPR/Cas9 technology has proven to be a valuable system to generate genetargeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5%(19/29) of pregnant ewes and 41.5%(22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock. © 2015 Crispo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


PubMed | Institute Reproduccion Animal Uruguay, French Institute of Health and Medical Research and Institute Pasteur Of Montevideo
Type: Journal Article | Journal: PloS one | Year: 2015

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.


Garcia-Pintos C.,Institute Reproduccion Animal Uruguay | Menchaca A.,Institute Reproduccion Animal Uruguay
Small Ruminant Research | Year: 2016

The objective of this study was to evaluate the ovarian response to equine chorionic gonadotropin (eCG) administrated during the luteal phase after insemination in sheep. Thirty cyclic multiparous ewes received a fixed-time intrauterine insemination (Day 0) and were assigned to three experimental groups that received 400 IU of eCG on Day 5 (n = 10) or Day 10 (n = 10), while another group remained as control group without eCG (n = 10). Corpus luteum area and follicular diameter were daily measured by transrectal ultrasonography and serum progesterone concentrations were daily determined by radioimmunoassay during 30 days after insemination in pregnant ewes or until the following ovulation in non pregnant ewes. The administration of eCG on Day 10, but not on Day 5, increased corpus luteum size and serum progesterone concentrations (P < 0.05). Follicular dynamics were influenced by both eCG treatments on Day 5 and Day 10, promoting the growth of the largest follicle present during treatment or increasing the length of the wave that emerged after treatment (P < 0.05). In conclusion, the administration of 400 IU of eCG 10 days after insemination in sheep increased corpus luteum size and progesterone concentrations, as well as promoted follicular development. This information could be useful to develop new pharmacological strategies to improve the establishment of pregnancy in sheep. © 2016 Elsevier B.V..

Loading Institute Reproduccion Animal Uruguay collaborators
Loading Institute Reproduccion Animal Uruguay collaborators