Donor Females Research Articles

Impact of litter size on the hematological and iron status of gilts, sows and newborn piglets: a comparative study of domestic pigs and wild boars

BackgroundThe critically low hepatic iron stores of newborn piglets are considered to be a major cause of neonatal iron deficiency in modern breeds of domestic pig (Sus domestica). The main factor believed to contribute to this phenomenon is large litter size, which has been an objective of selective breeding of pigs for decades. As consequence, iron transferred from the pregnant sow has to be distributed among a greater number of fetuses.ResultsHere, we investigated whether litter size influences red blood cell (RBC) indices and iron parameters in Polish Large White (PLW) piglets and gilts. Small and large litters were produced by the transfer of different numbers of embryos, derived from the same superovulated donor females, to recipient gilts. Piglets from large litters obtained following routine artificial insemination were also examined. Our results clearly demonstrated that varying the number of piglets in a litter did not affect the RBC and iron status of 1-day-old piglets, with all showing iron deficiency anemia. In contrast, gilts with small litters displayed higher RBC and iron parameters compared to mothers with large litters. A comparative analysis of the RBC status of wild boars (having less than half as many piglets per litter as domestic pigs) and PLW pigs, demonstrated higher RBC count, hemoglobin level and hematocrit value of both wild boar sows and piglets, even compared to small-litter PLW animals.ConclusionsThese findings provide evidence that RBC and iron status in newborn PLW piglets are not primarily determined by litter size, and indicate the need to study the efficiency of iron transport across the placenta in domestic pig and wild boar females.

Os embriões bovinos podem ser transferidos com sucesso após 40 anos de criopreservação?

ABSTRACT Global advances in reproductive biotechnology have allowed for the transfer of embryos from donor females with high genetic merit to recipients using the cryopreservation technique, which preserves an embryo of excellent quality and viability, thereby achieving a feasible pregnancy rate. The objective of this study was to evaluate the quality and viability of Holstein embryos that have been cryopreserved for more than 40 years under glycerol freezing. The embryos were transferred to the recipient heifers using a non-surgical method. Two 17-month-old Holstein heifers (360 kg live weights) which were clinically healthy and reproductively active were used as the recipients. Two bovine embryos of Grade 1 quality were thawed and evaluated for their morphology. Of the two embryo transfers, one pregnancy was achieved, resulting in the birth of a calf. Therefore, embryos frozen in liquid nitrogen and glycerol as a cryopreservative for more than 40 years maintained their quality and viability to produce a live calf.

Stratified haploid identification system through the R1-nj kernel and reduced seedling vigor in tropical maize germplasm

Abstract. Thawarorit A, Dermail A, Lertrat K, Chankaew S, Suriharn K. 2023. Stratified haploid identification system through the R1-nj kernel and reduced seedling vigor in tropical maize germplasm. Biodiversitas 24: 4262-4268. Haploid identification through the R1-nj marker is commonly applied in in-vivo doubled haploid technology in maize, although misclassification issues often occur. We explored the innate properties of maternal haploids at the seedling stage to verify true haploids and to reduce false positives. There are many questions about whether reduced seedling vigor is effective for haploid verification in tropical maize backgrounds. This study aimed to evaluate the haploid induction rate of Stock6-derived F3 population inducers and to investigate the effectiveness of reduced seedling vigor at the V2/V3stage as an alternative marker to verify putative haploids. Field trials and haploid induction were conducted in the rainy season of 2021 and the dry season of 2021/22 in Khon Kaen, Thailand. Two tropical source germplasm: P789 and S7328, were included as donor females. Haploids were identified through the R1-nj marker and reduced seedling vigor. Significant reduction of haploid frequency up to 2.5% on average between the two identification methods, indicating that a considerable number of false positives could be found using reduced seedling vigor. Inducer population K11 was promising since it performed a stable ability to produce haploids over donors and seasons and had a low misclassification rate. Further breeding strategies and haploid selection schemes are discussed.

In vitro and in vivo embryo production efficiency in Flemish and Holstein donor females.

The aim of this study was to compare embryo production efficiency in Flemish and Holstein donor females using ovum pick-up and in vitro fertilization (OPU-IVF) or in vivo production (superovulation; SOV) procedures. The study was conducted using a split-plot design, with eight Flemish and eight Holstein non-lactating cycling females. Females were subjected to ten weekly OPU/IVF sessions and/or two SOV/embryo collections sessions at a 63-day interval, for a total of 160 OPU-IVF and 32 SOV sessions. Mean numbers of follicles and corpora lutea, and cumulus-oocyte complex (COC) recovery rates were similar between breeds after the OPU and SOV sessions. However, Flemish donors yielded better quality grade II COCs (301, 41.9%) than Holstein females (609, and 202, 33.1%). Also, cleavage and blastocyst rates, and the total number and the mean number of viable embryos obtained after OPU-IVF were higher in Flemish (49.6% and 11.8%, and 63 and 11.8 per donor, respectively) than in Holstein (32.8% and 7.2%, and 34 and 7.2 per donor, respectively) females. Flemish females were also more efficient in yielding viable embryos after SOV (111, 7.3 per donor) than Holstein (48, 3.3 per donor) females. Overall, Flemish donor females had better responses to OPU-IVF or SOV procedures than Holstein counterparts. Irrespective of the breeds, SOV procedures were more efficient than OPU-IVF in yielding more viable embryos, under the conditions of this study. Both reproductive procedures were useful tools for the genetic conservation of the Flemish cattle breed in Southern Brazil.

Anti-Müllerian Hormone and Its Utility in Cattle Reproduction

Reproductive biotechnologies offer us greater possibilities to improve animal genetics. However, the success of these depends on different factors such as the proper selection of the donor female. For this reason, endocrine markers have been used to evaluate the ovarian reserve, which allow a successful selection of donors. Recent research has shown, among other things, that concentrations greater than 0.130 ng/mL of anti-Mullerian hormone (also known as Muller-inhibiting substance, which is a member of the transforming growth factor beta superfamily of growth and differentiation factors) are related to donors of more than fifteen transferable embryos. Therefore, this review describes studies showing that the measurement of anti-Müllerian hormone concentrations, before superovulation programs, reduces the costs per embryo produced.

The Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana for Management of the Melon Fly Zeugodacus cucurbitae: Pathogenicity, Horizontal Transmission, and Compatability with Cuelure

Simple SummaryThe melon fly Zeugodacus cucurbitae is an invasive fruit fly that causes extensive damage to many fruit crops. Sustainable management of this pest can be achieved through environmentally friendly and safely integrated pest management (IPM), in which the use of biopesticides is a key component. In this study, various isolates of entomopathogenic fungi were evaluated on two developmental stages of this pest. The fungal isolate ICIPE 69 has the greatest potential. The study also evaluated whether ICIPE 69 could be used together with the male pheromone of the melon fruit fly, as well as whether infected flies could contaminate clean flies. Our study indicates that ICIPE 69 can be included in IPM packages to reduce yield losses.In the laboratory, the pathogenicity of thirteen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin against the melon fly Zeugodacus cucurbitae (Coquillett) were assessed by exposing adults to 0.3 g of dry conidia (~3 × 109 conidia) of each isolate for 5 min and monitoring mortality for up to 5 days. Compatibility with a male pheromone, cuelure, (4-(p-acetoxyphenyl)-2-butanone), was determined by testing conidial germination and germ tube growth of the most promising isolate, M. anisopliae ICIPE 69, in the presence of cuelure at different temperatures. For horizontal transmission, the flies were separated by sex, separately exposed to M. anisopliae ICIPE 69, and subsequently mixed with non-exposed flies from the other sex. The most pathogenic isolates were M. anisopliae ICIPE 69, 18, and 30, causing mortalities of 94, 87, and 81%, with 5 days post-exposure, respectively. Metarhizium anisopliae ICIPE 69 caused the highest pupal mortality of 74%, with 15 days post-exposure. Horizontal transmission of M. anisopliae ICIPE 69 among male and female Z. cucurbitae was confirmed by 59 and 67% mortality after exposure to infected donor males and females, respectively. Metarhizium anisopliae ICIPE 69 affected the oviposition, but not hatchability, of infected Z. cucurbitae females. Metarhizium anisopliae ICIPE 69 is, therefore, a potential isolate for biopesticide development for Z. cucurbitae management in cucurbit production systems.

Fetal resorption coincides with dysregulated LH secretion in AMH-overexpressing mice

Female anti-Müllerian hormone (AMH) overexpressing (Thy1.2-AMHTg/0) mice experience fetal resorption (miscarriage) by mid-gestation. This study examined whether the ovary, uterine implantation sites and hypothalamus are potential sites of AMH action, as AMH type-2 receptor (AMHR2) expression is reported in each tissue. Pregnancy in Thy1.2-AMHTg/0 mice was compared to wild-type (WT) mice via histological examination of implantation sites, hormone assays, embryo culture and embryo transfer. Uterine AMH and AMHR2 expression was examined by RT-qPCR and immunohistochemistry. The first signs of fetal resorption in the Thy1.2-AMHTg/0 dams occurred at embryonic day 9.5 (E9.5) with 100% of fetuses resorbing by E13.5. Cultured embryos from Thy1.2-AMHTg/0 dams had largely normal developmental rates but a small proportion experienced a minor developmental delay relative to embryos from WT dams. However, embryos transferred from WT donor females always failed to survive to term when transferred into Thy1.2-AMHTg/0 dams. Amh and Amhr2 mRNA was detected in the gravid uterus but at very low levels relative to expression in the ovaries. Progesterone and estradiol levels were not significantly different between WT and Thy1.2-AMHTg/0 dams during pregnancy but luteinizing hormone (LH) levels were significantly elevated in Thy1.2-AMHTg/0 dams at E9.5 and E13.5 relative to WT dams. Collectively, these experiments suggest that AMH overexpression does not cause fetal resorption through an effect on oocytes or preimplantation embryo development. The Thy1.2-AMHTg/0 fetal resorption phenotype is nearly identical to that of transgenic LH overexpression models, suggesting that neuroendocrine mechanisms may be involved in the cause of the miscarriage.

Effect of DHA on the quality of In vitro produced bovine embryos

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that improves fertility by increasing membrane fluidity. Moreover, embryos produced by donor females supplied with n-3 PUFA did not show any difference in terms of the lipid profile after 7 days of culture. The present study aimed to investigate the effects of DHA (20 and 100 μM) coupled with carnosine (5 mg/mL), an antioxidant, during oocyte maturation and embryo development on the developmental and cryosurvival rates and the number of pluripotent cells. Free fatty acid receptor-4 (FFAR4), which is able to bind DHA, was visualised by immunostaining. The addition of DHA in the in vitro development (IVD) medium decreased the percentage of pluripotent SOX2 positive cells compared with the control (8.4% vs. 10.9%) without affecting the number of cells (196.7 vs. 191.6 cells) or the developmental (20.9% vs. 23.9% blastocysts rate on D7) and cryosurvival rates (86.3% vs 86.2%). Such a decrease in pluripotent cells, relevant to the differentiation of the first lineage within the inner cell mass, represents an improvement in the embryo quality. On the contrary, embryos without any pluripotent SOX2-positive cells would not be able to achieve gestation. Future studies should follow up these results by carrying out embryo transfers to assess the beneficial effects of DHA supplementation.

Factors affecting outcomes of embryo transfer in dromedary camels: A retrospective study.

The present study aimed to use a comprehensive approach for evaluating 12 factors related to embryo quality (shape, size and transparency), donors (age, ET type, number of recovered embryos and day of uterine flushing), recipients (age), males (age and individual variations) and environment (season and year) which could affect the outcome of ET in terms of pregnancy (PR) and pregnancy losses in dromedary camels. During three breeding seasons, 116 donor females were mated repeatedly at 12- to 14-day intervals by fertile male camels (n=33) without stimulation of the ovaries (WSPO). Superovulation (SPO ET) regimen was applied for each donor female twice or thrice per season. In the occasions of applying superovulation regimen, donor females having an ovulatory follicle were mated instead of GnRH administration and superovulation regimen was applied 4days post-mating (MIX ET). The uteri of all donor females were flushed at Day 8 or 9 post-mating, and a total of 2,095 embryos were recovered and transferred individually to 924 recipient females. Pregnancy diagnoses were conducted at Day 10 after ET (Days 18-19 of gestation) by using progesterone assay and by transrectal ultrasonography (TRU) at Days 30 and 60 of gestation. By using logistic regression analysis, transparency of embryos and age of recipient females had significant effects on PR at Days 18-19 (p<.01), 30 (p<.01) and 60 (p<.01; p<.05, respectively) of gestation. The shape of embryos had significant effects on the PR at Days 30 (p<.05) and 60 (p<.01) of gestation. Type of ET and the breeding season (year) had significant (p<.05) effects on the PR at Day 30, while day of flushing had the same effect on PR at Day 60. Regarding the pregnancy losses, transparency and shape of the embryo, type of ET, breeding season had significant (p<.05) effect on the late embryonic mortalities (LEM) and shape and season of year had significant (p<.01 and p<.05, respectively) effect on LEM/early foetal mortalities (EFM). Regarding male individual factor, there was a tendency for a significant (p=.055) effect of male camels on the PR at Days 18-19 and rate of LEM. In conclusion, transferring a spherical, transparent or a large-sized embryo (>750µm) into recipient females ageing between 8 and 11 y could greatly improve the PR from Days 18 to 60 of gestation. Also, embryo recovered from donor females with Mix ET type or embryos sired by certain male camel or at Day 8 post-mating of the donor could improve the 2-month PR. In addition, transferring a transparent or spherical-shaped embryo or embryos recovered from donor females with SPO or Mix ET could reduce the pregnancy losses during the first 2months of pregnancy.

Ovarian superstimulation with FSH in Wagyu breed bovine donors impacts folicular dynamics but does not improve the amount of embryos

The increase of the world population generates the need to raise the production of food of vegetal and animal origin. In Brazil, livestock farming has evolved, demonstrating the important role of the country in the production of food. In this way, researchers of this productive chain have been looking for technologies related to production and reproduction, above all, to the use of reproduction biotechnologies, seeking to increase the production of different bovine breeds. Thus, the objective of this research was to investigate the efficacy of ovarian FSH super stimulation in bovine Wagyu oocyte donor females on follicular dynamics and in vitro embryo production. Twelve Wagyu animals, aged 12 to 24 months, randomly distributed in a crossover design were used in two groups: Group 1= animals not stimulated with FSH and Group 2= animals stimulated with FSH. The follicular, oocyte and embryonic variables were evaluated. It was observed that ovarian overstimulation in Wagyu oocyte donor cows with FSH improved the mean and large follicles but reduced the rate of oocyte recovery and, despite the best percentage of viable oocytes, there was no improvement in the amount of embryos produced in vitro.

Development of a GnRH-PGF2α Based Synchronization and Superstimulation Protocol for Fixed-Time Mating in Llama Embryo Donors.

The aim of the present study was to evaluate the application of a GnRH-PGF2α based synchronization and superstimulation protocol for fixed-time natural mating in llama embryo donors. All females (n = 8) received 8 μg IM of GnRH analog (GnRHa; buserelin) on day 0, regardless of follicular status. After eight days, another GnRHa dose was administered followed by 250 μg IM PGF2α (cloprostenol). A dose of 1000 IU IM of equine chorionic gonadotrophin (eCG) was applied on day 12 and a new dose of PGF2α was administered on day 13. All embryo donors were mated with a male of proven fertility followed by a GnRHa dose on day 18. 24 h later, mating was repeated with a different male. Transcervical uterine flushing for embryo recovery was carried out on all females on day 26. Recipient females received one dose of GnRHa (day 0) two days after the first mating of embryo donor females. A 75% (6/8) of embryo donors responded to the superstimulation treatment with a range of 2 to 5 corpus luteums (CLs) on embryo recovery day. A total of 24 CLs were registered, with a mean of 4 ± 0.9 CLs per female. Embryo recovery rate was 66.7% (16/24), with a range of 0 to 4 embryos and a mean of 2.7 ± 1.5 embryos per female. Regarding quality of the recovered embryos, 56.2% were grade I, 6.2% were grade II and 37.5% were grade V (untransferable; arrested morulae). Grade I and II embryos (n = 10) were transcervically transferred into recipient females (n = 10) six days after inducing their ovulation. At 24 days after embryo transfer (ET), a 50% pregnancy rate was registered. In conclusion, a group of llama embryo donors can be synchronized and superstimulated using a fixed-time mating protocol based on GnRHa, PGF2α, and eCG without the necessity of using ultrasonography in the field.

Electroporation and genetic supply of Cas9 increase the generation efficiency of CRISPR/Cas9 knock-in alleles in C57BL/6J mouse zygotes

CRISPR/Cas9 machinery delivered as ribonucleoprotein (RNP) to the zygote has become a standard tool for the development of genetically modified mouse models. In recent years, a number of reports have demonstrated the effective delivery of CRISPR/Cas9 machinery via zygote electroporation as an alternative to the conventional delivery method of microinjection. In this study, we have performed side-by-side comparisons of the two RNP delivery methods across multiple gene loci and conclude that electroporation compares very favourably with conventional pronuclear microinjection, and report an improvement in mutagenesis efficiency when delivering CRISPR via electroporation for the generation of simple knock-in alleles using single-stranded oligodeoxynucleotide (ssODN) repair templates. In addition, we show that the efficiency of knock-in mutagenesis can be further increased by electroporation of embryos derived from Cas9-expressing donor females. The maternal supply of Cas9 to the zygote avoids the necessity to deliver the relatively large Cas9 protein, and high efficiency generation of both indel and knock-in allele can be achieved by electroporation of small single-guide RNAs and ssODN repair templates alone. Furthermore, electroporation, compared to microinjection, results in a higher rate of embryo survival and development. The method thus has the potential to reduce the number of animals used in the production of genetically modified mouse models.

C57BL/6J and B6129F1 Embryo Transfer: Unilateral and Bilateral Transfer, Embryo Number and Recipient Female Background Control for the Optimization of Embryo Survival and Litter Size

Simple SummaryEmbryo transfer is a common procedure in rodent facilities related to rederivation protocols, recovery of cryopreserved embryos and production of genetically engineered animals. This procedure consists of the transfer of mouse embryos into the oviduct of a pseudopregnant recipient female in order to obtain live pups. The aim of this study is to further characterize the optimal conditions to perform embryo transfer using wild type strains and particularly the bilateral transfer. C57BL/6J and B6129F1 embryos were freshly collected and transferred to recipient females, after overnight culture to a 2-cell stage and tested for different conditions (unilateral and bilateral surgical procedures, variable number of embryos and reciprocity between recipient mother and embryo’s genetic background). The results achieved show that C57BL/6J transfers with a low number of embryos provide higher success rates when using unilateral transfers, but for bilateral transfers a minimum number of embryos seems to be necessary. B6129F1 presented similar results, but bilateral transfers were more effective with low number of embryos. These results allow a better planning of the embryo transfer procedure, considering low number of embryos and the choice of unilateral transfers as the ideal condition for an optimal outcome. This optimization has a positive impact on the 3R’s application: it can help to reduce the number of recipient and donor females and to improve recipient female’s welfare through the use of a less invasive technique.Embryo transfer (ET) is a common procedure in rodent facilities. Optimizing this technique may help to reduce the number of animals, but little information is available regarding wild type strains and the conditions that affect embryo transfer. To explore this theme, 2-cell C57BL/6J embryos were transferred after overnight culture of freshly collected zygotes using different conditions: unilateral transfers using a total of 6, 8, 12, 15, 20 and 25 embryos were performed initially; then, this strain was also used for bilateral transfers using a total of 6, 12 and 20 embryos equally divided by the two oviducts. Groups of 25 embryos were not tested for the bilateral technique, since this condition produced the lower success rate when using the unilateral technique and 20 embryos would still represent a large number of embryos. A group of 2-cell B6129F1 embryos was also transferred using unilateral and bilateral ET with 6, 12 and 20 embryos. Crl:CD1(ICR) were used as recipient females for non-reciprocal transfers and C57BL/6J were used to test reciprocal transfers (only tested for six C57BL/6J unilateral transfers). Unilateral transfers using C57BL/6J mice produced higher success rates using six embryos, compared to the other groups transferred unilaterally (p-values between 0.0001 and 0.0267), but the mean number of pups per litter was not different among groups. Bilateral transfer produced higher number of pups when 20 embryos were divided by the two oviducts compared to six (p = 0.0012) or 12 (p = 0.0148) embryos, but with no differences in success rates. No statistical differences were found between the groups of B6129F1, but better results were obtained on bilateral transfers using a total of six embryos. For the strain tested (C57BL/6J), the uterine environment (Crl:CD1(ICR) or C57BL/6J recipient) does not impact the outcome of the technique. These results complement previous work published using genetically engineered mice strains and show that unilateral transfers using low number of embryos (6), produce better outcomes when compared to bilateral or unilateral transfers using more embryos. It also highlights differences between the outcome of bilateral transfers in the two strains tested. A set of historical data of genetically engineered mice at a C57BL/6J background was also included, confirming that lower embryo numbers are related to higher success rates. Together, the outcome of these experiments can be important to reduce the number of recipient and donor females, optimize embryo transfers and improve animal welfare discouraging the use of a more invasive technique.

Metarhizium anisopliae and Beauveria bassiana: Pathogenicity, Horizontal Transmission, and Their Effects on Reproductive Potential of Thaumatotibia leucotreta (Lepidoptera: Tortricidae).

The polyphagous moth, Thaumatotibia leucotreta (Meyrick) is among the major constraints to the horticultural industry in East Africa. In a search of sustainable control methods, the pathogenicity of the dry conidia of 17 Metarhizium anisopliae (Metschn.) and five Beauveria bassiana (Bals.) (Vuill.) isolates were tested against T. leucotreta moths. The conidial uptake by a single moth, horizontal transmission, and effect of fungal inoculant on egg-laying and hatchability for the pest were also assessed. The findings from this study showed that tested fungi were virulent to the moths, with 12 isolates causing mortality that ranged between 58.8 and 94.2% for M. anisopliae and three isolates between 57.6 and 84.6% for B. bassiana. The entomopathogenic fungi isolates, M. anisopliae ICIPE 69 and B. bassiana ICIPE 279 were highly virulent with low lethal times to 50% mortality (LT50) of 3.81 and 5.13 d, respectively. The isolate, ICIPE 69 conidia acquisition by a single moth were approximately 4.58 × 106 and 3.51 × 106 for males and females, respectively. The lowest survival rate (3.33 %) caused by inoculum transmission was recorded in donor males compared to 16.67% in recipient males. Fecundity was reduced by 33.6 and 25.9% for donor and recipient females, respectively. The pest can acquire conidia from the treated substrate and transmission is then possible between adults. ICIPE 69 is virulent and reduces the reproduction potential of the pest after transmission. The findings are discussed in the prospects of microbial control of this pest using an autodissemination technique.

Aspiration oocyte recovery by laparoscopy for embryo transfer in goats: a review

The aspiration of oocytes by laparoscopy for the transfer of embryos is a technique used in the extraction of oocytes from a genetically superior animal, with the purpose of producing in vitro embryos to implant them in a female, which allows to increase the response to selection in a program of genetic improvement, by obtaining animals above the population average and because the generation interval can be reduced. Prior to the aspiration of the oocytes, it is necessary to prepare the donor and recipient females based on exogenous hormones, to achieve maximum production and extraction of oocytes in the same donor female and, consequently, a greater probable number of embryos produced and transferred. With the aim of describing the different protocols used in the aspiration of oocytes by the laparoscopic technique for the transfer of embryos in goats, the present literature review was carried out through scientific publications.

Supplementation with sunflower seeds in beef cattle did not impact on oocyte and in vitro embryo production.

Supplementation with compounds rich in linoleic acid, including sunflower seed supplementation, promotes increase in conception rates in cows. We aimed to evaluate whether the sunflower seed (linoleic acid source) supplementation in beef donor females alters the plasma concentrations of cholesterol, triglycerides, HDL and LDL, increases the number and quality of oocytes, increases the cleavage rates and determines an improvement in number and quality of in vitro produced blastocysts. Thus, Nelore females were divided into two groups of 15 animals to receive supplementation with or without sunflower seed for 57days. Females underwent follicular aspiration and the oocytes were subjected to in vitro embryo production. There was no difference (p>.1) between control group and group supplemented with sunflower seed on the number of displayed follicles; number of aspired oocytes; recovery rate; cleavage rate; number of embryos; number of blastocysts; embryos number of grades I and II; plasma concentrations of total cholesterol, triglycerides; HDL and LDL. Therefore, sunflower seed supplementation in oocyte donors did not increase the number and quality of oocytes, cleavage rates and the number and quality of blastocysts produced in vitro.

Nutritional impact on gene expression and competence of oocytes used to support embryo development and livebirth by cloning procedures in goats

Changes in the nutritional plan have been shown to affect oocyte quality, crucial to oocyte donors animals used in cloning. This study aimed to evaluate the impact of diets with increasing nutritional levels (maintenance diet=M; 1.3M; 1.6M; 1.9M) fed to goats for four weeks on follicular fluid composition, gene expression and oocyte competence used to cloning in goats. Donor females were superovulated for the retrieval of matured oocytes and physical measurements reported. After four weeks, groups receiving diets above maintenance increased thickness of subcutaneous adipose tissue and body weight, with higher values in 1.9M Group (P<0.05). Treatments did not affect follicular density, number of aspirated follicles, retrieved and matured oocytes. Animals from 1.3M group had lower (P<0.05) maturation rate (44.0%) and number of viable oocytes (65.3%) than M (68.8%) and 1.9M (76.0%). Follicular fluid glucose concentrations increased with nutritional levels (P=0.010), with a difference (P<0.05) between groups 1.9M (11.4±2.6mg/dL) and M (2.6±0.5mg/dL). The diet did not affect the expression of GDF9, BMP15, and BAX genes in oocytes, but BCL2 and apoptotic index were significantly higher (P<0.05) in the 1.3M and 1.6M groups than the other groups. Following the transfer of cloned embryos, one fetus was born live of a twin pregnancy in the 1.9M Group. The association between energy intake and oocyte quality suggests better nutritional use by oocytes when the maximum flow was used (1.9M), but the optimal feeding level in cloning still needs refinement.

157 EFFECT OF FSH STARVATION (COASTING) FOLLOWING SUPEROVULATION ON OOCYTE COMPETENCE AND CLONING EFFICIENCY IN GOATS

Oocyte competence plays a key role in the overall efficiency of reproductive biotechnologies. In cattle, FSH starvation following superovulation (coasting) improves oocyte competence, blastocyst yield and pregnancy outcome when used in ovum pickup-in vitro production programs. The aim of this study was to compare the effect of coasting after exogenous FSH stimulation on goat oocyte quality and competence to support in vitro maturation and in vivo embryo development following cloning procedures in goats. Donor and recipient preparation, cumulus-oocyte complex (COC) retrieval and selection, IVM, cloning by somatic cell nuclear transfer, embryo transfer, and pregnancy diagnosis (Days 23–26) were performed according to our established procedures [Martins et al. 2016 doi: 10.1089/cell.2015.0082]. Cumulus-oocyte complexes were obtained in vivo from 71 cycling FSH-stimulated mature Nubian-crossed goats, combined or not with FSH starvation (coasting period). Donor females were oestrous synchronized with a progesterone intravaginal insert (Day 0). On Day 10, a 0.75-mg D-cloprostenol dose was given IM, with the onset of the superovulation treatment, composed of five 20-mg FSH doses (Folltropin®, Bioniche Animal Health, Pullman, WA, USA), via IM at 12-h intervals. Donors were subjected to laparoscopic ovum pickup either 9 h (control group, n = 36) or 21 h (coasting group, n = 35) after the last FSH dose, respectively. Skin fibroblast cell cultures from a male neonate were co-transfected with a mammary gland expression vector with the human lactoferrin (hLF) coding sequence and with CRISPR/Cas9 system either for the PRNP prion gene or the Rosa26 locus. A bi-allelic hLF-PRNP and a mono-allelic hLF-Rosa26 cell colony were used for cloning. Data were compared by ANOVA or the χ2 test (P &lt; 0.05). No differences were observed between control and coasting for number of follicles (18.7 ± 1.4 v. 21.2 ± 1.7), and retrieved (17.3 ± 1.2 v. 20.7 ± 1.9), viable (15.9 ± 1.1 v. 19.6 ± 1.8), Grade I (1.5 ± 0.3 v. 2.5 ± 0.5), and Grades III+IV (6.0 ± 0.6 v. 5.7 ± 0.7) COC, as well as for COC retrieval (92.4%, 574/621 v. 94.5%, 685/725) and fusion (62.8%, 273/435 v. 61.3%, 311/507) rates, respectively, irrespective of the cell lines. However, the coasting group rendered higher number of Grade II COC (11.3 ± 1.2 v. 8.4 ± 0.7), number and proportion of Grades I+II COC (13.9 ± 1.5 v. 9.9 ± 0.9, 70.8% v. 62.4%), and maturation rate (70.9% v. 65.4%) than the control group, respectively, for a lower proportion of Grades III+IV (29.2% v. 37.6%, respectively). A total of 213 and 233 Day-1 cloned embryos from the control and the coasting groups were transferred to 18 (96/9 hLF-PRNP and 117/9 hLF-Rosa26 cells) and 19 (128/11 hLF-PRNP and 105/8 hLF-Rosa26 cells) female recipients, respectively, resulting in 1/9 (11.1%) and 4/11 (36.4%) pregnancies from the hLF-PRNP cells, and 3/9 (33.3%) and 3/8 (37.5%) from the hLF-Rosa26 cells, for the control (4/18, 22.2%) and coasting (7/19, 36.8%) groups, respectively, for an overall pregnancy rate of 29.7% (11/37). In conclusion, the use of coasting improved oocyte quality and in vitro maturation rate, also appearing to increase pregnancy outcome after goat cloning.

Generation of transgenic marmosets expressing genetically encoded calcium indicators.

Chronic monitoring of neuronal activity in the living brain with optical imaging techniques became feasible owing to the continued development of genetically encoded calcium indicators (GECIs). Here we report for the first time the successful generation of transgenic marmosets (Callithrix jacchus), an important nonhuman primate model in neurophysiological research, which were engineered to express the green fluorescent protein (GFP)-based family of GECIs, GCaMP, under control of either the CMV or the hSyn promoter. High titer lentiviral vectors were produced, and injected into embryos collected from donor females. The infected embryos were then transferred to recipient females. Eight transgenic animals were born and shown to have stable and functional GCaMP expression in several different tissues. Germline transmission of the transgene was confirmed in embryos generated from two of the founder transgenic marmosets that reached sexual maturity. These embryos were implanted into six recipient females, three of which became pregnant and are in advanced stages of gestation. We believe these transgenic marmosets will be invaluable non-human primate models in neuroscience, allowing chronic in vivo monitoring of neural activity with functional confocal and multi-photon optical microscopy imaging of intracellular calcium dynamics.

Prepubertal heifers versus cows—The differences in the follicular environment

The oocyte quality is to a large extent influenced by the sexual maturity of the donor female. Although this phenomenon has already been broadly described in domestic animals, the underlying mechanisms are poorly understood. Published data focus on oocyte ultrastructure, fertilization abnormalities, and blastocyst developmental rate. The goal of the present experiment was to characterize the follicular environment (oocyte, cumulus [CC] and granulosa (GC) cells as well as follicular fluid [FF]) in ovarian follicles of prepubertal heifers and cows. Each experimental replicate included the following set of traits within individual follicles: lipid droplets (LDs) number in oocytes, expression of seven genes involved in energy metabolism (fatty acids [FAs] metabolism—ELOVL2, ELOVL5, SCD, FADS2, glucose transport—GLUT1, GLUT3, GLUT8) in CC and GC as well as FA composition and glucose concentration in FF. According to our results, cow oocytes were larger in diameter and contained more LD than those from prepubertal heifers, both before and after IVM. The LD number was also higher in cow oocytes after IVM, when compared to immature oocytes. The FF from cow follicles had elevated glucose content similarly to the majority of the analyzed FA. Transcript analysis revealed differences for five out of seven analyzed genes (ELOVL, FADS2, SCD, GLUT3, GLUT8) in CC and GC cells. However after considering the female category, the only difference was noticed for the mRNA of SCD gene, which was more abundant in cow GC. This finding may indicate distinct roles of CC and GC in follicular energy metabolism. In conclusions, we suggest that distinct properties of follicular environment in prepubertal heifers and cows may be responsible for differences in the quality of oocytes from the two categories of donors. We hypothesize that suboptimal environment in heifer follicles (glucose and FA lower content in FF) determines reduced quality of their oocytes (lower diameter and LD number) and limited maturation potential. Besides, energy demands of heifer oocytes may be restricted due to a low LD number, exerting a negative effect on the development of the future embryo. The advantages of cow gametes (e.g., higher LD number and diameter) attributed to oocytes of superior quality may support the statement that cows donate oocytes of better quality than heifers.