Refinements in embryo manipulation applied to CRISPR technology in livestock.

The implementation of CRISPR technology in large animals requires further improvements in embryo manipulation and transfer to be applied with commercial purposes. In this study we report (a) developmental competence of CRISPR/Cas microinjected zygotes subjected to in vitro culture in large scale programs in sheep; (b) pregnancy outcomes after early-stage (2-8-cell) embryo transfer into the oviduct or the uterine horn; and (c) embryo survival and birth rate after vitrification/warming of CRISPR/Cas microinjected zygotes. Experiment 1 consisted of a retrospective analysis to evaluate embryo developmental rate of in vitro produced zygotes subjected to CRISPR/Cas microinjection (n = 7,819) compared with a subset of non-microinjected zygotes (n = 701). Development rates to blastocyst on Day 6 were 20.0% for microinjected zygotes and 44.9% for non-injected zygotes (P < 0.05). In Experiment 2, CRISPR/Cas microinjected zygotes were transferred on Day 2 after in vitro fertilization (2-8 cell embryos) into the oviductal ampulla (n = 262) or into the uterine horn (n = 276) in synchronized recipient ewes at prefixed time (i.e., approximately two days after ovulation). Pregnant/transferred recipients (24.0% vs. 25.0%), embryo survival/transferred embryos (6.9% vs. 6.2%), and born lambs/pregnant embryos (72.2% vs. 100.0%) did not differ significantly in the two groups. In Experiment 3, CRISPR/Cas microinjected zygotes were maintained under in vitro culture until blastocyst stage (Day 6), and subjected to vitrification/warming via the Cryotop method (n = 474), while a subset of embryos were left fresh as control group (n = 75). Embryos were transferred into the uterine horn of recipient females at prefixed time 8.5 days after the estrous synchronization treatment (i.e., approximately six days after ovulation). Pregnancy rate (30.8% vs. 48.0%), embryo survival rate (14.8% vs. 21.3%), and birth rate (85.7% vs. 75.0%) were not different (PNS) between vitrified and fresh embryos, respectively. In conclusion, the current study in sheep embryos reports (a) suitable developmental rate after CRISPR/Cas microinjection (i.e., 20%), even though it was lower than non-microinjected zygotes; (b) similar outcomes when Day 2-embryos were placed into the uterine horn instead of the oviduct, avoiding both time-consuming and invasive oviduct manipulation, and extended in vitro culture during one week; (c) promising pregnancy and birth rates obtained with vitrification of CRISPR/Cas microinjected embryos. This knowledge on in vitro embryo development, timing of embryo transfer, and cryopreservation of CRISPR/Cas microinjected zygotes have practical implications for the implementation of genome editing technology in large animals.

Ivermectin reduces in vivo coronavirus infection in a mouse experimental model.

The objective of this study was to test the effectiveness of ivermectin for the treatment of mouse hepatitis virus (MHV), a type 2 family RNA coronavirus similar to SARS-CoV-2. Female BALB/cJ mice were infected with 6,000 PFU of MHV-A59 (group infected, n = 20) or infected and then immediately treated with a single dose of 500 µg/kg ivermectin (group infected + IVM, n = 20) or were not infected and treated with PBS (control group, n = 16). Five days after infection/treatment, the mice were euthanized and the tissues were sampled to assess their general health status and infection levels. Overall, the results demonstrated that viral infection induced typical MHV-caused disease, with the livers showing severe hepatocellular necrosis surrounded by a severe lymphoplasmacytic inflammatory infiltration associated with a high hepatic viral load (52,158 AU), while mice treated with ivermectin showed a better health status with a lower viral load (23,192 AU; p < 0.05), with only a few having histopathological liver damage (p < 0.05). No significant differences were found between the group infected + IVM and control group mice (P = NS). Furthermore, serum transaminase levels (aspartate aminotransferase and alanine aminotransferase) were significantly lower in the treated mice than in the infected animals. In conclusion, ivermectin diminished the MHV viral load and disease in the mice, being a useful model for further understanding this therapy against coronavirus diseases.

Ovarian superstimulatory response and embryo development using a new recombinant glycoprotein with eCG-like activity in mice.

Pharmacological ovarian control required for the implementation of artificial insemination and embryo-related technologies usually includes the use of eCG, naturally produced in pregnant mares. In this study, we report the superovulatory response and embryo development in mice obtained with a new glycoprotein with eCG-like activity (reCG) produced by recombinant DNA technology. A total of 150 females from three different mouse strains (C57BL/6J, BALB/cJ and B6D2F1/J) were subjected to a superstimulatory protocol consisting of 5 IU of natural eCG (n = 50), 5 IU of reCG (n = 50), or received a placebo (no-eCG, n = 50) by intraperitoneal route, followed by 5 IU of human chorionic gonadotropin 48 h later. Overall, no significant differences were observed in the total number of ova/zygotes (33.6 ± 2.4 vs 28.7 ± 2.6; P = NS) and viable ova/zygotes (31.5 ± 2.4 vs 25.8 ± 2.5; P = NS) collected per female among eCG and reCG treated females, respectively, which were greater (P < 0.05) than those obtained in no-eCG treated females (6.9 ± 0.7 and 5.9 ± 0.7, respectively). Zygotes derived from the three experimental groups (n = 2914) were subjected to in vitro culture until hatching 4.5 days post coitum (dpc). Regardless of the mouse strain, no differences were observed among eCG and reCG treated females for overall cleavage rate 1.5 dpc (58.5% vs 60.5%), development rate 3.5 dpc (47.2% vs 48.9%) and hatching rate 4.5 dpc (49.5% vs 54.5) (P = NS). Control females from no-eCG treated group showed lower cleavage and development rates (36.4% and 29.7%, respectively; P < 0.05). In conclusion, this study reports for the first time comparable superovulatory response and embryo development between recombinant and natural eCG treatment, which has important implications for reproductive technologies in several species.

Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair.

Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.

Local influence of the corpus luteum on the ipsilateral oviduct and early embryo development in the ewe.

The objective of this study was to evaluate the local effect of the corpus luteum (CL) on ipsilateral oviduct-uterus functionality and early embryo development in ewes. A total of 499 embryos were transferred on Day 1 after in vitro fertilization into the ipsilateral (n = 250) and contralateral oviducts (n = 249) of 13 ewes on Day 1 after ovulation (18-20 embryos per oviduct). On Day 6, their reproductive tracts were collected and their uterine horns were flushed for embryo recovery. More recovered embryos, a higher proportion of blastocysts, and more viable embryos were collected when the embryos were transferred into the ipsilateral oviducts (P < 0.05). In addition, almost five times higher P4 concentrations and significantly lower E2 concentrations, with higher P4:E2 ratio, were found in the ipsilateral than contralateral oviductal tissue (P < 0.05). Furthermore, a higher concentration of adiponectin was found in the ipsilateral uterine tissue macerates than in the contralateral side to the CL. The ipsilateral oviductal tissue had a lower expression of PGR and IGFBP5, but the transcript expression of ADIPOR1 was higher in the ipsilateral oviductal tissue. In the uterus, the mRNA expression of ESR1, IGFBP3, IGFBP5, and LEPR was higher or tended to be higher in the ipsilateral than contralateral uterine tissue. Uterine flushing fluid collected from the ipsilateral uterine horn had lower insulin concentrations than the contralateral horn, while no differences were found in the P4 and E2 concentrations. In conclusion, on Day 6 post-ovulation, P4 was elevated in the ipsilateral oviductal tissue, embryo development was advanced, and differential gene expression of PGR, ESR1, IGFBP3, IGFBP5, LEPR, and ADIPOR1 in the oviductal or uterine tissue was found between the ipsilateral and contralateral side. This study demonstrates local regulation of the ovary on the ipsilateral oviduct/uterine horn in the ewe.

CRISPR in livestock: From editing to printing.

Precise genome editing of large animals applied to livestock and biomedicine is nowadays possible since the CRISPR revolution. This review summarizes the latest advances and the main technical issues that determine the success of this technology. The pathway from editing to printing, from engineering the genome to achieving the desired animals, does not always imply an easy, fast and safe journey. When applied in large animals, CRISPR involves time- and cost-consuming projects, and it is mandatory not only to choose the best approach for genome editing, but also for embryo production, zygote microinjection or electroporation, cryopreservation and embryo transfer. The main technical refinements and most frequent questions to improve this disruptive biotechnology in large animals are presented. In addition, we discuss some CRISPR applications to enhance livestock production in the context of a growing global demand of food, in terms of increasing efficiency, reducing the impact of farming on the environment, enhancing pest control, animal welfare and health. The challenge is no longer technical. Controversies and consensus, opportunities and threats, benefits and risks, ethics and science should be reconsidered to enter into the CRISPR era.

Cumulus cells during in vitro fertilization and oocyte vitrification in sheep: Remove, maintain or add?

The objective was to evaluate the developmental competence of immature and matured ovine oocytes after removing, maintaining or adding cumulus cells (CC) associated to vitrification by Cryotop method. Three experiments were performed involving 3,144 oocytes. In Experiment 1, CC were removed from immature, matured or fertilized oocytes subjected to in vitro embryo production. In Experiment 2, oocytes were vitrified either in MI or MII stage with or without CC, while a control group with CC remained unvitrified. In Experiment 3, oocytes partially denuded from CC were vitrified either in MI or MII stage, and a co-culture of fresh CC was added or not soon after warming to complete in vitro maturation (IVM) and in vitro fertilization (IVF), or IVF, respectively, while a control group remained unvitrified. In Experiment 1, the cleavage rate, development rate on Day 6 and blastocyst rate on Day 8 were improved when CC were maintained until the end of IVF (P < 0.05). In Experiment 2, vitrification of oocytes with enclosed CC showed a tendency to increase cleavage (P = 0.06) and improved blastocyst rate (P < 0.05). In Experiment 3, adding CC as co-culture after vitrification-warming tended to improve cleavage rate (P = 0.06) and increased hatching rate (P < 0.05). Regarding oocyte stage, vitrification of in vitro matured oocytes resulted in greater developmental competence than immature stages (P < 0.05). In conclusion, CC seems to have a relevant role for in vitro embryo development in either fresh or vitrified oocytes.

Genotypic Characterization of Emerging Avian Reovirus Genetic Variants in California.

This study focuses on virus isolation of avian reoviruses from a tenosynovitis outbreak between September 2015 and June 2018, the molecular characterization of selected isolates based on partial S1 gene sequences, and the full genome characterization of seven isolates. A total of 265 reoviruses were detected and isolated, 83.3% from tendons and joints, 12.3% from the heart and 3.7% from intestines. Eighty five out of the 150 (56.6%) selected viruses for sequencing and characterization were successfully detected, amplified and sequenced. The characterized reoviruses grouped in six distinct genotypic clusters (GC1 to GC6). The most represented clusters were GC1 (51.8%) and GC6 (24.7%), followed by GC2 (12.9%) and GC4 (7.2%), and less frequent GC5 (2.4%) and GC3 (1.2%). A shift on cluster representation throughout time occurred. A reduction of GC1 and an increase of GC6 classified strains was noticed. The highest homologies to S1133 reovirus strain were detected in GC1 (~77%) while GC2 to GC6 homologies ranged between 58.5 and 54.1%. Over time these homologies have been maintained. Seven selected isolates were full genome sequenced. Results indicated that the L3, S1 and M2 genes, coding for proteins located in the virus capsid accounted for most of the variability of these viruses. The information generated in the present study helps the understanding of the epidemiology of reoviruses in California. In addition, provides insights on how other genes that are not commonly studied add variability to the reovirus genome.

Serum progesterone concentrations during FSH superstimulation of the first follicular wave affect embryo production in sheep.

The aim of the present study was to evaluate the effect of serum progesterone concentrations during the superstimulatory treatment of the first follicular wave on fertilization rate and embryo development in sheep. A total of 71 Merino ewes received a superstimulatory FSH treatment during Wave 1 of ovarian follicular development (Day 0 Protocol), which was administrated under low progesterone concentrations typical of the early luteal phase (control group, n = 33) or under high progesterone concentrations induced by the administration of an intravaginal device from Day 0 to Day 3 containing 0.3 g progesterone (n = 38). Intrauterine insemination after FSH superstimulation was followed by uterine flushing 6 days later. Serum progesterone concentrations from Day 0 to 3 were greater in those ewes treated with progesterone (P < 0.05), while serum estradiol-17ß concentrations were not affected by the treatment. Although the mean number of corpora lutea per donor was not affected by the progesterone treatment, the number of collected ova and embryos was greater in progesterone treated than untreated ewes (6.6 ± 0.7 compared with 4.6 ± 0.9, respectively; P < 0.05). Furthermore, progesterone treatment increased fertilization rate (93.3% compared with 83.3%; P < 0.05) and the proportion of Grade 1 embryos (67.7% compared with 52.7%; P < 0.05) compared with the control group. In conclusion, oocyte fertilization rate and embryo quality are improved by high progesterone concentrations during FSH superstimulation, which suggests an important role of progesterone during preovulatory follicular development.

Oocyte developmental competence is improved by relatively greater circulating progesterone concentrations during preovulatory follicular growth.

This study evaluated the effect of progesterone priming during follicular growth on oocyte competence to undergo oocyte cleavage and embryo development in sheep. Two experiments were performed on a total of 195 females that either received or did not receive a progesterone treatment (CIDR-type device) during the first follicular wave, beginning soon after ovulation (i.e., Day 0 of the experiment). On Day 3, the follicular population and oocyte quality (Experiment 1 and 2) and the competence of oocytes for cleavage and embryo development (Experiment 2) were evaluated after laparoscopic ovum pickup (LOPU) and in vitro fertilization. In Experiment 1, in a 2 × 2 factorial study the progesterone priming treatment (treated or not) was or was not associated with a single dose of FSH in a slow-release hyaluronic acid preparation given on Day 0. The follicular population on Day 3 and the number and morphology of recovered cumulus oocyte complexes (COCs) were not affected by the progesterone treatment (P = NS) but were improved by the FSH administration (P <  0.05). An interaction between both treatments was observed (P <  0.05), with more desirable outcome with the females that received both the progesterone and the FSH treatments. In Experiment 2, half of the females received the exogenous progesterone priming, and all females received FSH on Day 0. After follicular aspiration on Day 3, the cleavage rate and the embryo development rate following in vitro fertilization and culture were greater in those females that received the progesterone treatment (P <  0.05). In conclusion, these studies provide evidence that progesterone treatment during follicular growth affects oocyte competence, with the greater progesterone concentrations enhancing the oocyte's capacity to undergo cleavage and embryo development.

Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos.

The objective was to evaluate pregnancy outcomes and birth rate of in vivo derived vs. in vitro produced ovine embryos submitted to different cryopreservation methods. A total of 197 in vivo and 240 in vitro produced embryos were cryopreserved either by conventional freezing, or by vitrification with Cryotop or Spatula MVD methods on Day 6 after insemination/fertilization. After thawing/warming and transfer, embryo survival rate on Day 30 of gestation was affected by the source of the embryos (in vivo 53.3%, in vitro 20.8%; P < 0.05) and by the method of cryopreservation (conventional freezing 26.5%, Cryotop 52.0%, Spatula MVD 22.2%; P < 0.05). For in vivo derived embryos, survival rate after embryo transfer was 45.6% for conventional freezing, 67.1% for Cryotop, and 40.4% for Spatula MVD. For in vitro produced embryos, survival rate was 7.3% for conventional freezing, 38.7% for Cryotop, and 11.4% for Spatula MVD. Fetal loss from Day 30 to birth showed a tendency to be greater for in vitro (15.0%) rather than for in vivo produced embryos (5.7%), and was not affected by the cryopreservation method. Gestation length, weight at birth and lamb survival rate after birth were not affected by the source of the embryo, the cryopreservation method or stage of development (average: 150.5 ± 1.8 days; 4232.8 ± 102.8 g; 85.4%; respectively). This study demonstrates that embryo survival and birth rate of both in vivo and in vitro produced ovine embryos are improved by vitrification with the minimum volume Cryotop method.

RAPID COMMUNICATION: Nerve growth factor influences cleavage rate and embryo development in sheep.

Recent information about Nerve growth factor (NGF), a protein traditionally associated to the nervous system that regulates survival and maturation of developing neurons, suggests that it may exert action also on different levels in the reproductive system. The aim of this study was to evaluate the effect of NGF added during in vitro oocyte maturation, fertilization or in vitro embryo development in sheep. Nerve growth factor was supplemented to the culture medium at 0, 100, or 1,000 ng/mL, during either in vitro maturation (Exp. 1), in vitro fertilization (Exp. 2), or in vitro culture (Exp. 3). In addition, NGF mRNA expression was determined in cumulus cells and oocytes. Nerve growth factor induced early cleavage when added during oocyte maturation or fertilization, improved embryo development when added during fertilization, and had no significant effect when added during embryo culture. In general, the effect was more evident with 100 rather than 1,000 ng/mL (P < 0.05). Expression of endogenous NGF was not detected in oocytes, and increased in cumulus cells when 1,000 ng/mL of NGF was added during fertilization, but not during maturation and embryo culture. In conclusion, the addition of NGF during oocyte maturation and fertilization affects in vitro cleavage and embryo development in sheep. We suggest a possible effect of this growth factor on oocyte maturation and mainly on the fertilization process.

New insights and current tools for genetically engineered (GE) sheep and goats.

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 people's 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 people's life quality around the world.

Myopathy of the Pipping Muscles, Hepatosis Dietetica, and Cataracts in Emu Chicks (Dromaius Novaehollandiae).

Seven emu chicks (Dromaius novaehollandiae) from a farm with poor hatchability (16-18%) and increased neonatal mortality were presented for necropsy with a history of death at or within a few days after hatching. Macroscopic examination revealed subcutaneous edema and hemorrhages and swelling of the pipping muscles in the proximal neck (71%), pale liver with hemorrhages (71%), noninternalized residual yolk sac (86%) and anasarca (14%). Histologically, the most remarkable findings were necrosis of the musculus complexus (100%) of the pipping muscles, as well as myocardial necrosis and mineralization (29%). Liver contained severe multifocal hepatocellular necrosis and hemorrhages (57%), and both eyes exhibited swollen and vacuolated lenticular fibers in 5 chicks (100%) in which the eyes were examined. The lesions observed here are suggestive of a nutritional deficiency. The deficiency was confirmed by finding low levels of vitamin E in the liver, and vitamin E and vitamin A levels in the feed.

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes.

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.

Cryotolerance of Day 2 or Day 6 in vitro produced ovine embryos after vitrification by Cryotop or Spatula methods.

This study was conducted to evaluate the cryotolerance of in vitro produced ovine embryos submitted to vitrification at different developmental stages using two methods of minimum volume and rapid cooling rate. Embryos were vitrified at early stage (2 to 8-cells) on Day 2 or at advanced stage (morulae and blastocysts) on Day 6 after in vitro fertilization. Vitrification procedure consisted of the Cryotop (Day 2, n=165; Day 6, n=174) or the Spatula method (Day 2, n=165; Day 6, n=175). Non vitrified embryos were maintained in in vitro culture as a control group (n=408). Embryo survival was determined at 3h and 24h after warming, development and hatching rates were evaluated on Day 6 and Day 8 after fertilization, and total cell number was determined on expanded blastocysts. Embryo survival at 24h after warming increased as the developmental stage progressed (P<0.05) and was not affected by the vitrification method. The ability for hatching of survived embryos was not affected by the stage of the embryos at vitrification or by the vitrification method. Thus, the proportion of hatching from vitrified embryos was determined by the survival rate and was lower for Day 2 than Day 6 vitrified embryos. The percentage of blastocysts on Day 8 was lower for the embryos vitrified on Day 2 than Day 6 (P<0.05), and was lower for both days of vitrification than for non-vitrified embryos (P<0.05). No interaction of embryo stage by vitrification method was found (P=NS) and no significant difference was found in the blastocyst cell number among vitrified and non-vitrified embryos. In conclusion, both methods using minimum volume and ultra-rapid cooling rate allow acceptable survival and development rates in Day 2 and Day 6 in vitro produced embryos in sheep. Even though early stage embryos showed lower cryotolerance, those embryos that survive the vitrification-warming process show high development and hatching rates, similar to vitrification of morulae or blastocysts.

Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.

Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4%; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3%, 46/117) and IVF embryos (39.6%, 44/111). Pregnancy rate was detected in 50.0% (6/12) of recipient ewes with TG embryos, in 46.7% (7/15) with IVF embryos, and in 65.0% (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9%) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs.

New approaches to superovulation and embryo transfer in small ruminants.

The present paper reviews the current state of knowledge in multiple ovulation and embryo transfer (MOET) technology in small ruminants, focusing on recently reported information. Major new findings are related to follicular wave patterns in small ruminants, the elucidation of follicular dominance and the integration of this information into ovarian superstimulatory treatment protocols. Follicular dynamics determine steroid and gonadotrophin secretion, follicular responses to FSH, ovulatory responses and embryo yield. Protocols that control follicular dominance have been designed to allow the initiation of superstimulation at the beginning of a follicular wave. New approaches consist of (1) synchronisation of ovulation for superstimulation during Wave 1 (i.e. Day 0 protocol), (2) pretreatment with a gonadotrophin-releasing hormone (GnRH) antagonist from 10 days prior to FSH treatment to avoid follicular dominance and (3) progesterone-oestradiol cotreatment to synchronise follicle wave emergence. These protocols provide a homogeneous pool of small follicles that are gonadotrophin responsive, enhancing the superovulatory response and embryo yield with a reduction in the incidence of unovulated follicles and early regression of corpora lutea. In addition, the rate of fertilisation failure has been reduced by using an inducer of ovulation (i.e. GnRH) associated with intrauterine insemination. In summary, the application of recently acquired knowledge has resulted in relevant improvements in MOET programmes in small ruminants.

Day 0 protocol: superstimulatory treatment initiated in the absence of a large follicle improves ovarian response and embryo yield in goats.

A new superstimulatory protocol (Day 0 Protocol) to initiate FSH treatment in the absence of a large follicle was compared to a traditional protocol in goats. The Day 0 Protocol (n=44) consisted of pre-treatment with progesterone and eCG to synchronize ovulation and the emergence of Wave 1, with FSH starting 84 h after the end of progesterone exposure (i.e., soon after ovulation). The traditional protocol (n=46) consisted of 11 d of progesterone exposure, with FSH treatment beginning 2 d before the end of progesterone exposure. Treatment with FSH was initiated in the absence of a large follicle in 37/44 and in 6/46 goats in the Day 0 Protocol and traditional protocol, respectively (P<0.01). There was more CL in the Day 0 Protocol than in the traditional protocol (breeding season: 9.6+/-0.6 and 6.3+/-0.8, P<0.05; non-breeding season: 14.3+/-1.5 and 10.7+/-1.5; P<0.05). More Grades 1 and 2 embryos were recovered in the Day 0 Protocol than in the traditional protocol (breeding season: 4.8+/-0.7 and 1.8+/-0.5, P<0.05; non-breeding season: 5.6+/-1.1 and 3.5+/-0.7, P=0.07). Similarly, the proportion of embryos that were Grades 1 and 2 was higher for the Day 0 Protocol than for the traditional protocol (breeding season: 81/114, 71%, versus 16/43, 37%, P<0.05; non-breeding season: 118/203, 58% versus 95/205, 46%, P<0.05). In summary, the Day 0 Protocol, was effective in initiating superstimulatory treatment in the absence of a large follicle, and compared to the traditional protocol, induced a higher ovulation rate and better embryo yield in goats.