Melatonin enhances in vitro developmental competence of cumulus-oocyte complexes collected by ovum pick-up in prepubertal and adult dairy cattle.

Bovine oocytes from prepubertal donors have been used for in vitro embryo production to decrease the generation interval. However, reduced cumulus-oocyte competence, mainly attributed to increased apoptosis, has been observed in oocytes/embryos collected from prepubertal donors. Here, we investigated the effects of the potent antioxidative molecule melatonin on cumulus-oocyte competence and embryo development in prepubertal and adult dairy cattle in vitro. A total of fifteen Holstein Friesian calves, six to ten months old (7.6 ± 1.34 months of age). And fifteen adult cows with one to four calvings (2.3 ± 0.96 calvings) were enrolled as ovum pick up (OPU) donors in this study. Cumulus-oocyte complexes (COCs) were cultured either in the presence or absence of melatonin (0.01 nM). The proportion of cleavage stages, blastocysts, and advanced blastocysts was determined. Embryo quality was assessed via differential staining to determine the total embryonic cells and allocation to the inner cell mass (ICM) and trophectoderm (TE) cells. Melatonin treatment yielded a greater percentage of blastocysts compared to the control group, i.e. oocytes from both adult cows (P = 0.0485; 24.8 ± 3.5% vs. 16.0 ± 3.4%, respectively), and from prepubertal donors (P = 0.0007; Melatonin 23.1 ± 5.1% vs. Control: 11.1 ± 3.5%). Adult cows had significantly (P = 0.0370) greater advanced blastocyst rates than those found in the prepubertal group (13.9%± vs. 7.0±%, respectively). Additionally, the number of ICM, total cells, and the ratios ICM: Total, ICM: TE, respectively, were greater (P < 0.05) after melatonin treatment compared with the control group (39.1 ± 2.8, 98.6 ± 5.7, 0.4 ± 0.01, and 0.7 ± 0.04 vs. 27.3 ± 2.9, 81.2 ± 5.8, 0.34 ± 0.01, and 0.52 ± 0.04, respectively). Blastocysts derived from adult cows had a greater number of TE (P = 0.01) and total embryonic cells (P = 0.0095) compared to the prepubertal donor group (63.5 ± 3.2 and 101.05 ± 4.8 vs. 48.9 ± 4.3 and 78.8 ± 6.5, respectively). Nevertheless, embryonic cell counting in embryos derived from prepubertal COCs equated to that observed from adult donors after melatonin exposure. In conclusion, these results indicate that the presence of melatonin during in vitro maturation improves cumulus-oocyte competence, embryo development, and quality by increasing the allocation of embryonic cells to the ICM compartment and the total number of embryonic cells in both adult and prepubertal bovine donors.

CRISPR/Cas12a mediated knock-in of the Polled Celtic variant to produce a polled genotype in dairy cattle.

In modern livestock farming horned cattle pose an increased risk of injury for each other as well as for the farmers. Dehorning without anesthesia is associated with stress and pain for the calves and raises concerns regarding animal welfare. Naturally occurring structural variants causing polledness are known for most beef cattle but are rare within the dairy cattle population. The most common structural variant in beef cattle consists of a 202 base pair insertion-deletion (Polled Celtic variant). For the generation of polled offspring from a horned Holstein-Friesian bull, we isolated the Polled Celtic variant from the genome of an Angus cow and integrated it into the genome of fibroblasts taken from the horned bull using the CRISPR/Cas12a system (formerly Cpf1). Modified fibroblasts served as donor cells for somatic cell nuclear transfer and reconstructed embryos were transferred into synchronized recipients. One resulting pregnancy was terminated on day 90 of gestation for the examination of the fetus. Macroscopic and histological analyses proved a polled phenotype. The remaining pregnancy was carried to term and delivered one calf with a polled phenotype which died shortly after birth. In conclusion, we successfully demonstrated the practical application of CRISPR/Cas12a in farm animal breeding and husbandry.

Possible detrimental effects of beta-2-microglobulin knockout in pigs.

BACKGROUND: Despite major improvements in pig-to-primate xenotransplantation, long-term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits. METHODS: In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta-2-microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M-KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules. RESULTS: Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4 weeks due to unexpected disease processes. CONCLUSION: Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta-2-microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future.

Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes.

BACKGROUND: Xenotransplantation is considered to be a promising solution to the growing demand for suitable donor organs for transplantation. Despite tremendous progress in the generation of pigs with multiple genetic modifications thought to be necessary to overcoming the severe rejection responses after pig-to-non-human primate xenotransplantation, the production of knockout pigs by somatic cell nuclear transfer (SCNT) is still an inefficient process. Producing genetically modified pigs by intracytoplasmic microinjection of porcine zygotes is an alluring alternative. The porcine GGTA1 gene encodes for the α1,3-galactosyltransferase that synthesizes the Gal epitopes on porcine cells which constitute the major antigen in a xenotransplantation setting. GGTA1-KO pigs have successfully been produced by transfecting somatic cells with zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or CRISPR/Cas targeting GGTA1, followed by SCNT. METHODS: Here, we microinjected a CRISPR/Cas9 vector coding for a single-guide RNA (sgRNA) targeting exon 8 of the GGTA1 gene into the cytoplasm of 97 in vivo-derived porcine zygotes and transferred 86 of the microinjected embryos into three hormonally synchronized recipients. Fetuses and piglets were analyzed by flow cytometry for remaining Gal epitopes. DNA was sequenced to detect mutations at the GGTA1 locus. RESULTS: Two of the recipients remained pregnant as determined by ultrasound scanning on day 25 of gestation. One pregnancy was terminated on day 26, and six healthy fetuses were recovered. The second pregnancy was allowed to go to term and resulted in the birth of six healthy piglets. Flow cytometry analysis revealed the absence of Gal epitopes in four of six fetuses (66%), indicating a biallelic KO of GGTA1. Additionally, three of the six live-born piglets (50%) did not express Gal epitopes on their cell surface. Two fetuses and two piglets showed a mosaicism with a mixed population of Gal-free and Gal-expressing cells. Only a single piglet did not have any genomic modifications. Genomic sequencing revealed indel formation at the GGTA1 locus ranging from +17 bp to -20 bp. CONCLUSIONS: These results demonstrate the efficacy of CRISPR/Cas to generate genetic modifications in pigs by simplified technology, such as intracytoplasmic microinjection into zygotes, which would significantly facilitate the production of genetically modified pigs suitable for xenotransplantation. Importantly, this simplified injection protocol avoids the penetration of the vulnerable pronuclear membrane, and is thus compatible with higher survival rates of microinjected embryos, which in turn facilitates production of genetically modified piglets.

DNA methylation and mRNA expression of developmentally important genes in bovine oocytes collected from donors of different age categories.

Epigenetic changes are critical for the acquisition of developmental potential by oocytes and embryos, yet these changes may be sensitive to maternal ageing. Here, we investigated the impact of maternal ageing on DNA methylation and mRNA expression in a panel of eight genes that are critically involved in oocyte and embryo development. Bovine oocytes were collected from donors of three different age categories-prepubertal (9-12 months old), mature (3-7 years old), and aged (8-11 years old)-and were analyzed for gene-specific DNA methylation (bTERF2, bREC8, bBCL-XL, bPISD, bBUB1, bDNMT3Lo, bH19, and bSNRPN) and mRNA expression (bTERF2, bBCL-XL, bPISD, and bBUB1). A total of 1,044 alleles with 88,740 CpGs were amplified and sequenced from 362 bovine oocytes. Most of the detected molecules were either fully methylated or completely unmethylated. Only 9 out of 1,044 alleles (<1%) were abnormally methylated (>50% of CpGs with an aberrant methylation status), and seven of the nine abnormally methylated alleles were within only two candidate genes (bDNMT3Lo and bH19). No significant differences were detected with regard to mRNA expression between oocytes from the three groups of donors. These results suggest that genes predominantly important for early embryo development (bH19 and bDNMT3Lo) are less resistant to abnormal methylation than genes critically involved in oocyte development (bTERF2, bBCL-XL, bPISD, bBUB1, and bSNRPN). Establishment of DNA methylation in bovine oocytes seems to be largely resistant to changes caused by maternal ageing, irrespective of whether the genes are critical to achieve developmental competence in oocytes or early embryos. Mol. Reprod. Dev. 83: 802-814, 2016 © 2016 Wiley Periodicals, Inc.

Oocyte pre-IVM with caffeine improves bovine embryo survival after vitrification.

Cryopreservation of in vitro produced bovine embryos is associated with significantly reduced survival rates, mainly due to insufficient quality of the embryos. Caffeine supplementation during IVM has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-IVM with caffeine on oocyte maturation, intraoocyte cAMP concentration, developmental competence after IVF, and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2 hours culture before IVM with or without caffeine (0, 1, 5, 10, 20, 30 mM), or standard IVM (no pre-IVM). Oocytes were in vitro matured and fertilized and zygotes were cultured under standard in vitro conditions until Day 8. Expanded blastocysts derived from either standard control or the 10-mM caffeine treatments were submitted to vitrification. Caffeine delayed meiotic resumption after 9-hour IVM in a concentration-dependent manner. The cAMP levels were similar before and after IVM. Matured oocytes, cleavage, and blastocyst rates were reduced in the 30-mM caffeine concentration and were similar among the other treatment groups. Number and proportion of inner cell mass and trophectoderm cells in blastocysts did not differ among treatments. Forty-eight hours after thawing, hatching rates were higher in the 10-mM caffeine group (73.8%) compared with the standard control (59.7%). Reexpansion rates and total number of cells after 48 hours were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation before IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance.

Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle.

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24 h IVM/control), cAMP30 (2 h pre-IVM (forskolin-IBMX), 30 h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.

Risk of infection after iatrogenic perforation of the gut wall? Evaluation of preventive strategies in a randomized controlled animal trial.

BACKGROUND: Interventional endoscopies entail a risk of infection secondary to perforation of the luminal wall. Thereby, bacteria may be introduced into the sterile environment of the peritoneal cavity (PC). Limited data are available regarding the efficacy of prophylactic anti-infective treatments. The aim of the study was to examine the efficacy/safety of anti-infective means in the prevention of infection by interventional endoscopies in a randomized controlled animal trial. METHODS: Forty pigs were randomized to: 1: control; 2: oral lavage; 3: gastric lavage; 4: oral/gastric lavage; 5: i.m. antibiotics. Lavage was performed with Octenisept prior to the operation. After gastric wall perforation, peritoneoscopy was performed. Before the procedure, after closure and prior to autopsy, intraabdominal lavage for bacterial culture was taken using mini-laparoscopy. At autopsy, macroscopic appearance of the PC was scored. Lavage fluids were grown to identify/quantify bacterial load. Concentration of intraperitoneal bacteria at autopsy was defined as main outcome parameter. RESULTS: No major complications occurred in any of the procedures. Bacterial load of the PC at autopsy was significantly reduced with antibiotics compared to all other groups, whereas it did not differ between the lavage groups and control. Macroscopic scoring of the PC showed significant lower rate of intraabdominal abscesses in the antibiotic group compared to the lavage groups and control (p < 0.01). CONCLUSION: Only antibiotic prophylaxis is effective for the prevention of infection after iatrogenic perforation of the gastrointestinal wall. There was no difference between any form of lavage and the control group. Further studies in humans are required to prove these animal data.

Effects of long-term dietary supplementation with conjugated linoleic acid on bovine oocyte lipid profile.

Nutritional and environmental conditions around conception and during early embryonic development may have significant effects on health and well-being in adult life. Here, a bovine heifer model was used to investigate the effects of rumen-protected fat supplementation on oocyte quality and embryo development. Holstein-Friesian heifers (n=84) received a dietary supplement consisting of rumen-protected conjugated linoleic acid (CLA) or stearic acid (SA), each on top of an isocaloric basic diet. Oocytes were collected via ultrasound-guided follicular aspiration and subjected to in vitro maturation followed by either desorption electrospray ionisation mass spectrometry (DESI-MS) for lipid profiling of individual oocytes or in vitro fertilisation and embryo culture. The type of supplement significantly affected lipid profiles of in vitro-matured oocytes. Palmitic acid and plasmalogen species were more abundant in the mass spectra of in vitro-matured oocytes after rumen-protected SA supplementation when compared with those collected from animals supplemented with CLA. Lipid concentrations in blood and follicular fluid were significantly affected by both supplements. Results show that rumen-protected fatty-acid supplementation affects oocyte lipid content and may pave the way for the establishment of a large-animal model for studies towards a better understanding of reproductive disorders associated with nutritional impairments.

Endoscopic ultrasound-guided lymph node ablation with a novel radiofrequency ablation probe: feasibility study in an acute porcine model.

BACKGROUND AND STUDY AIMS: Radiofrequency ablation (RFA) is an accepted method of tissue destruction for solid organ tumors. Endoscopic ultrasound (EUS)-guided RFA has been used for lesions in the pancreas and liver, but there is limited experience of lymph node ablation using EUS-guided RFA. The aim of this study was to determine feasibility and safety of prototype EUS-guided RFA of mediastinal lymph nodes. METHODS: This was an endoscopic experimental feasibility study in a porcine model. After EUS-guided puncture of targeted lymph nodes, the stylet of a 19-G needle was replaced by a prototype RFA probe. RFA was performed by ERBE generator (bipolar settings: 10 watts, effect 2, 2 minutes). The animals were euthanized, and the targeted lymph nodes were identified and removed for histology and measurement of the effect achieved. RESULTS: A total of 18 mediastinal lymph nodes were ablated (mean size 20.8 ±â€Š6.6  mm in the long axis). The average length of exposed probe was 10.0 ± 3.0 mm. The mean length and diameter of necrosis was 9.8 ±â€Š3.6  mm and 5.5 ±â€Š1.6  mm, respectively. Linear regression comparing needle length with necrosis diameter revealed a coefficient gradient of r = 0.92 (P = 0.0001). With EUS-RFA a mean of 17.6 ±â€Š10.3 % (range 8.0 % - 53.2 %) of the respective lymph node area was ablated. No complications (i. e. hemodynamic instability, local bleeding, tissue damage) occurred during the procedure. Technical problems included stripping of the probe by the EUS needle and bending of the tip of the probe. CONCLUSIONS: EUS-RFA of lymph nodes was performed safely and successfully using a prototype EUS-compatible probe. This method may have the potential for future use in patient care.

A novel endoscopic prototype device for gastric full-thickness biopsy for the histopathologic diagnosis of GI neuromuscular pathology: in vivo porcine long-term survival study (with videos).

BACKGROUND: Many GI motility disorders are associated with underlying GI neuromuscular pathology, which requires full-thickness biopsies (FTB) for histopathologic diagnosis. Currently, none of the endoscopy-based attempts to obtain FTB specimens have proven suitable for routine use. This study evaluated a novel endoscopic prototype device (ED) for this purpose. OBJECTIVE: To determine (1) the ability of the ED to obtain suitable FTB specimens, (2) associated complications, (3) feasibility of reliable defect closure, and (4) ability to evaluate intramural neuromuscular components. DESIGN: Preclinical proof-of-concept study in 30 pigs. SETTING: Animal laboratory. INTERVENTION: Gastric FTB specimens were obtained with a circular cutter and anchor. The defect was closed by over-the-scope clips/T-tags. The resection site was inspected via laparoscopy. After 2 to 4 weeks, necropsy was carried out to evaluate late complications. MAIN OUTCOME MEASUREMENTS: Feasibility, safety, and closure rate of the procedure. FTB specimens were assessed by histology/immunohistochemistry to visualize enteric neuromusculature. RESULTS: A total of 29 of 30 procedures were successfully performed; one hemorrhage required endoscopic treatment. A total of 29 of 30 FTB specimens (mean diameter 9.1 mm) were retrieved in 7.1 ± 0.4 minutes (range 3.0-12.5 minutes), displaying optimal tissue quality. Defect closure took 10.8 ± 0.9 minutes (range 7.2-32 minutes). Laparoscopy did not reveal damage to adjacent organs. Necropsy showed well-healed scars at the resection site and no complications, peritonitis, or abscess formation. Histology showed smooth muscle layers and submucosal and myenteric ganglia. LIMITATIONS: Survival animal pilot study, no patients. CONCLUSION: The novel ED enabled safe harvesting of well-preserved FTB specimens. Defect closure proved to be reliable. All neuromuscular structures relevant for histopathologic evaluation of GI neuromuscular pathology were demonstrated. Further studies are needed to verify the efficacy of this prototype device in the entire gut and in humans.

In vivo oocyte developmental competence is reduced in lean but not in obese superovulated dairy cows after intraovarian administration of IGF1.

The present study investigated the role of IGF1 in lactating lean and non-lactating obese dairy cows by injecting 1 µg IGF1 into the ovaries prior to superovulation. This amount of IGF1 has been linked with pregnancy loss in women with the polycystic ovary syndrome (PCOS) and was associated with impaired bovine oocyte competence in vitro. Transcript abundance and protein expression of selected genes involved in apoptosis, glucose metabolism, and the IGF system were analyzed. Plasma concentrations of IGF1 and leptin, and IGF1 in uterine luminal fluid (ULF), were also measured. IGF1 treatment decreased embryo viability in lean cows to the levels observed in obese cows. Obese cows were not affected by IGF1 treatment and showed elevated levels of IGF1 (in both plasma and ULF) and leptin. Blastocysts from lean cows treated with IGF1 showed a higher abundance of SLC2A1 and IGFBP3 transcripts. IGF1 treatment reduced protein expression of tumor protein 53 in blastocysts of lean cows, whereas the opposite was observed in obese cows. IGF1 in plasma and ULF was correlated only in the control groups. Blastocyst transcript abundance of IGF1 receptor and IGFBP3 correlated positively with IGF1 concentrations in both plasma and ULF in lean cows. The detrimental microenvironment created by IGF1 injection in lean cows and the lack of effect in obese cows resemble to a certain extent the situation observed in PCOS patients, where IGF1 bioavailability is increased in normal-weight women but reduced in obese women, suggesting that this bovine model could be useful for studying IGF1 involvement in PCOS.

Clip closure versus endoscopic suturing versus thoracoscopic repair of an iatrogenic esophageal perforation: a randomized, comparative, long-term survival study in a porcine model (with videos).

BACKGROUND: Esophageal full-thickness wall repair is an important but unsolved issue in endoscopy. It is unknown how well endoscopic clip closure (ECC) and endoscopic closure with suturing (ECS) perform compared with the criterion standard of thoracoscopic closure (TC). OBJECTIVE: Comparison of technical success, feasibility, long-term patency, complications, and histological quality of the different closure techniques (ECC, ECS, TC) for esophageal perforations. DESIGN: Comparative animal study. SETTING: Approved animal facility. SUBJECTS: Eighteen pigs. INTERVENTIONS: Eighteen pigs were randomized, 6 each into 3 groups (ECC, ECS, TC). After endoscopic wall incision and mediastinoscopy, closure was performed by using 1 of the 3 techniques. After 8 to 12 weeks, pre-euthanasia endoscopic, necropsy, histological, and morphometric analyses were performed. MAIN OUTCOME MEASUREMENT: Long-term survival and histological quality of the repair. RESULTS: The closure of the esophageal incisions was successful in all pigs. On days 2 and 6, 1 animal died of mediastinitis, 1 in the ECS group because of reflux of gastric contents into the mediastinum before the repair and 1 in the TC group because of leakage of the sutured closure (P = 1.0). No strictures were seen on prenecropsy endoscopy. At necropsy, 1 mediastinal abscess was found in an ECS animal (P = 1.0). Minor complications included periesophageal adhesions and reactive lymph nodes in 3 of 6 (ECC group) and 5 of 6 (TC and ECS groups). Histology showed muscle layer defects up to 12 mm in width and 21 mm in length, with a trend toward smaller defect size of width and length in the ECS group of animals. LIMITATIONS: Animal study of limited size. CONCLUSIONS: Overall, ECS and ECC performed similarly to TC. ECS showed the smallest histological defects in the long-term repair.

Effects of eCG and FSH on ovarian response, recovery rate and number and quality of oocytes obtained by ovum pick-up in Holstein cows.

The goal of the present study was to compare the ovarian response, oocyte yields per animal, and the morphological quality of oocytes collected by ultrasound guided follicular aspiration from Holstein cows treated either with FSH or eCG. Twenty four normal cyclic, German Holstein cows were randomly divided into two groups. Fourteen cows received 3000 IU eCG on day-4 prior to ovum pick-up (OPU) (day 0), 2 days later (day-2), 625 microg cloprostenol was administered. On day-1 GnRH was administered i.m. and 24h later OPU (day 0) was performed. In ten cows a total dose of 500 IU follicle stimulating hormone (Pluset) was administered intramuscularly in a constant dosage for 4 days with intervals of 12h, starting on day-5. Luteolysis was induced by application of 625 microg cloprostenol on day-2. On day-1 (24h after the last FSH treatment) GnRH was administered i.m. and 24h later OPU (day 0) was performed. Ovarian follicles were visualized on the ultrasound monitor, counted and recorded. All visible antral follicles were punctured. Recovered oocytes were graded morphologically based on the cumulus investment. Average follicle number in ovaries was higher in FSH group than eCG group (p<0.05). Oocyte yields per animal did not differ between FSH and eCG groups. The proportion of grade A oocytes was higher in the FSH group in the than eCG group (p<0.05). Likewise, rate of grade C oocytes in FSH group were lower than eCG group (p<0.05). In conclusion, these results suggest that ovarian response, follicle number in ovaries and oocyte quality are affected by the type of gonadotropin and FSH is better alternative than eCG for OPU treatment.

Laparoscopical intrauterine insemination with different doses of fresh, conserved, and frozen semen for the production of ovine zygotes.

The objective of the present study was to increase the efficiency in the production of ovine zygotes suitable for microinjection via laparoscopical intrauterine insemination. In the first part of the study, 71 ewes of three different breeds were inseminated with one of two different insemination doses (50 x 10(6) or 300 x 10(6) sperm per inseminate) and semen was either freshly diluted, liquid conserved, or frozen/thawed, or females were mated by a fertile ram (controls). In the second part, a total of 46 ewes was inseminated with 300 x 10(6) freshly diluted sperm to verify the findings from part 1 and to unravel effects of breed and age of donor ewe. The oviducts were flushed 24-26 h after insemination and the success of insemination was assessed by microscopical examination. Recovery rates were 78.0+/-26.4 and 72.1+/-24.6% in parts 1 and 2 of the study, respectively. Of these oocytes 62.3 and 62.8% (parts 1 and 2, respectively) were fertilized. In part 1, the highest proportion (64.7%) of pronuclear stages was observed in the group inseminated with 300 x 10(6) freshly diluted semen and was significantly higher compared to the groups inseminated with 50 x 10(6) freshly diluted semen (25.5%, P<0.001), 300 x 10(6) liquid conserved semen (49.0%, P<0.001), or 50 x 10(6) frozen/thawed semen (39.6%, P<0.05). In the control group, the proportion of pronuclear stages amounted to 60.2%. Irrespective of the type of sperm conservation, the overall fertilization rate (zygotes plus 2-cell stages) was higher (P<0.05) following insemination with 300 x 10(6) sperm (68.2%) compared to 50 x 10(6) sperm (56.8%). In part 2, the proportion of pronuclear stages reached 54.2% with an overall fertilization rate of 62.9%. These results were affected by breed and age of the donor as crossbred and younger (<3 years) animals yielded the highest proportion of pronuclear stages. The present study shows that freshly diluted semen at a dosage of 300 x 10(6) sperm yields the highest fertilization rates, the greatest proportion of pronuclear stages and the lowest proportion of mature unfertilized oocytes. Further increases in yields of pronuclear stages can possibly be achieved by selection of sheep from the best suited breed and younger than 3 years of age.