Improving ovulation in gilts using anti-inhibin serum treatment combined with fixed-time artificial insemination.

For successful batch farrowing, porcine oestrus and ovulation must be synchronized using fixed-time artificial insemination (FTAI). However, exogenous gonadotropins, which are currently used in FTAI, negatively affect gilt ovulation. Here, we aimed to improve sexually mature gilt superovulation efficiency using passive immunization against inhibin during FTAI. Altrenogest-treated gilts were challenged with 10 ml anti-inhibin serum (AIS group, n = 6), 1,000 IU pregnant mare serum gonadotropin (PMSG group, n = 6), or 10 ml goat serum (control group, n = 6). Gilts in the AIS and PMSG groups were inseminated according to the FTAI protocol, and gilts in the control group were inseminated during natural oestrus. When PMSG was replaced by AIS during FTAI of gilts, ovulation rate and embryos recovered were significantly greater in the AIS group as compared to the other two groups (p < .05). Especially the average number of 6-8-cell embryos in the AIS group was significantly higher than that in the PMSG group (p < .01). Moreover, the blastocyst number in the AIS group was significantly higher than that in the PMSG group and the control group (p < .05). But there was no significant difference in the blastocyst number between the PMSG group and the control group (p > .05). Besides, plasma levels of estradiol-ß (E2) and progesterone (P4) were significantly greater in the AIS group as compared to the other two groups on Day 23 and D 27, respectively (p < .01). In summary, we devised an improved high-yield FTAI protocol for sexually mature gilts using AIS; this protocol had a greater superovulation efficiency than the FTAI using PMSG.

Isolation, characterization and germline chimera preparation of primordial germ cells from the Chinese Meiling chicken.

Primordial germ cells (PGCs) are precursors of germline cells that can generate sperm and eggs in adults, making them promising tools for transgenic animal preparation and germplasm preservation, especially in avians. In this study, we purified the PGCs from circulating embryonic blood of Chinese Meiling chickens using Nycodenz density centrifugation, and characterized them by alkaline phosphatase (AKP) staining, periodic acid-Schiff (PAS) staining and stage-specific embryonic antigen-1 (SSEA-1) immunostaining and PGC-specific gene amplification. The purified PGCs were also labeled with PKH26 and transferred into donor chicken embryos at the Hamburger-Hamilton (HH) stage 14 to 16, and cells with red fluorescence were observed in the gonads of 8-d-old embryos. When using about 200 PGCs isolated from Chinese Meiling chickens, microinjection into the dorsal aortas of recipient chickens with white feathers at stage HH14 to 16 resulted in germline chimeras that hatched and attained sexual maturity. The frequency of donor-derived yellow-feathered offspring from germline chimeric chickens was 12.6 ± 2.6% after mating with the white-feathered chickens. These results demonstrate that we had successfully purified the PGCs from the Chinese Meiling chicken. These germline cells could be used to preserve Chinese Meiling chickens.

Effect of shRNA-mediated Xist knockdown on the quality of porcine parthenogenetic embryos.

BACKGROUND: Parthenogenetically activated oocytes exhibit poor embryo development and lower total numbers of cells per blastocyst accompanied by abnormally increased expression of Xist, a long noncoding RNA that plays an important role in triggering X chromosome inactivation during embryogenesis. RESULTS: To investigate whether knockdown of Xist influences parthenogenetic development in pigs. We developed an anti-Xist short hairpin RNA (shRNA) vector, which can significantly inhibit Xist expression for at least seven days when injected at 12-13 hr after parthenogenetic activation. Embryonic cleavage, blastocyst formation, and total blastocyst cell numbers were compared during the blastocyst stage, as well as the expression of an X-linked gene and three pluripotent transcription factors. Knockdown of Xist significantly increases the total blastocyst cell number, but does not influence the rate of embryo cleavage and blastocyst formation. The expressions of Sox2, Nanog, and Oct4 were also significantly improved in the injected embryos compared with the control at the blastocyst stage, but the Foxp3 expression level was not increased significantly. CONCLUSIONS: The present study provides valuable information for understanding the role of Xist in parthenogenesis and presents a new approach for improving the quality of porcine parthenogenetic embryos. Developmental Dynamics 248:140-148, 2019. © 2018 Wiley Periodicals, Inc.

Non-invasive assessment of culture media from goat cloned embryos associated with subjective morphology by gas chromatography - mass spectroscopy-based metabolomic analysis.

Pre-implantation embryo metabolism demonstrates distinctive characteristics associated with the development potential of embryos. We aim to determine if metabolic differences correlate with embryo morphology. In this study, gas chromatography - mass spectroscopy (GC-MS)-based metabolomics was used to assess the culture media of goat cloned embryos collected from high-quality (HQ) and low-quality (LQ) groups based on morphology. Expression levels of amino acid transport genes were further examined by quantitative real-time PCR. Results showed that the HQ group presented higher percentages of blastocysts compared with the LQ counterparts (P < 0.05). Metabolic differences were also present between HQ and LQ groups. The culture media of the HQ group showed lower levels of valin, lysine, glutamine, mannose and acetol, and higher levels of glucose, phytosphingosine and phosphate than those of the LQ group. Additionally, expression levels of amino acid transport genes SLC1A5 and SLC3A2 were significantly lower in the HQ group than the LQ group (P < 0.05, respectively). To our knowledge, this is the first report which uses GC-MS to detect metabolic differences in goat cloned embryo culture media. The biochemical profiles may help to select the most in vitro viable embryos.

MBD1 and MeCP2 expression in embryos and placentas from transgenic cloned goats.

DNA methylation is an important form of epigenetic regulation in mammalian development. Methyl-CpG-binding domain protein 1 (MBD1) and methyl-CpG-binding domain protein 2 (MeCP2) are two members of the MBD subfamily of proteins that bind methylated CpG to maintain the silencing effect of DNA methylation. Given their important roles in linking DNA methylation with gene silencing, this study characterized the coordinated mRNA expression and protein localization of MBD1 and MeCP2 in embryos and placentas and aimed to analysis the effects of MBD1 and MeCP2 on transgenic cloned goats. Our result showed that MBD1 expression of transgenic cloned embryo increased significantly at the 2-4-cell and 8-16-cell stages (P < 0.05), then decreased at the morula and blastocyst stages (P < 0.05); MeCP2 expression in transgenic cloned embryo was significant decreased at the 2-4-cell stage and increased at the 8-16-cell stage (P < 0.05). Placenta morphology analysis showed that the cotyledon number of deceased transgenic cloned group (DTCG) was significantly lower than that the normal goats (NG) and in the live transgenic cloned goats (LTCG) (P < 0.05). MBD1 and MeCP2 were clearly detectable in the placental trophoblastic binucleate cells by immunohistochemical staining. Moreover, MBD1 and MeCP2 expression in DTCG was significant higher than in the NG and the LTCG (P < 0.05). In summary, aberrant expression of methylation CpG binding proteins MBD1 and MeCP2 was detected in embryonic and placental development, which reflected abnormal transcription regulation and DNA methylation involved in MBD1 and MeCP2. These findings have implications in understanding the low efficiency of transgenic cloning.

Age-associated and tissue-specific expression of osteopontin in male Hu sheep reproductive tract.

Osteopontin (OPN) is indispensable in mammalian reproduction, but the role of OPN in male reproductive tract and fertility remains unclear. The objective of this study is to elucidate the function of OPN by unveiling the localization and expression of OPN in the reproductive tract (testis, epididymis, and ductus deferens) of male Hu sheep in different ages (10-days, 4-months, and 8-months). To accomplish this, the localization, mRNA and protein expression patterns of OPN in all samples were investigated. Immune staining showed that OPN was present in the testicular interstitium of prepubertal Hu sheep testis (10-days and 4-months group), while it was immunostained in acrosomes of spermatids nearby adluminal compartment of seminiferous tubules in sexual maturity Hu sheep testis (8-months group). The localization of OPN in epididymis gradually changed from the loose connective tissue to the apical region of principal cells (pseudostratified columnar epithelium) with growing (10-days to 8-months). In addition, increase trend was observed in the mRNA expression levels of OPN with growing in the same reproductive tissues (P<0.05). Furthermore, two different OPN isoforms of 30kDa and 34kDa were detected in the reproductive tract of male Hu sheep by western blot. Immunofluorescence detection showed that OPN was localized in the cauda epididymal spermatozoa. These results suggested that the expression of OPN might be closely related to spermatogenesis and spermatozoa function in Hu sheep. This will be helpful for us to understand how OPN regulate the high reproductive capacity in Hu sheep.

Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system.

Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes.

Using cysteine/cystine to overcome oxidative stress in goat oocytes and embryos cultured in vitro.

Assisted reproductive techniques expose gametes to excessive concentrations of reactive oxygen species. The present study aimed to evaluate the effects of oxidative stress on apoptosis in goat oocytes and embryonic development. The results demonstrated that the addition of 100 µM hydrogen peroxide (H2O2) into media produces an oxidative environment during oocyte maturation. The number of cumulus cells positive for terminal deoxynucleotidyl transferase UTP nick end labeling, and the activity of caspase 3 in mature oocytes were increased, compared with the control group (P<0.05). In addition, the expression levels of mitochondrial regulators, including peroxisome proliferator­activated receptor Î³ coactivator-1 α (PGC-1α) and nuclear respiratory factor­1 (NRF­1) were increased in the oxidative oocytes, compared with those in the control group (P<0.05). The ratio of the proapoptotic gene, B cell lymphoma (Bcl-2)-associated X protein (BAX), to the anti­apoptotic gene, BCL­2, was higher in the H2O2 group, compared with the control group (P<0.05). To overcome oxidative stress in oocytes and embryos cultured in vitro, 200 µM cysteine and 200 µM cystine were added to the media, thereby increasing the concentration of intracellular glutathione (GSH) and assisting in maintaining the redox state of the cells. In conclusion, cysteine and cystine reduced the oxygen tension caused by H2O2, thereby providing a novel strategy for optimizing in vitro embryonic development systems.

Transgenesis of humanized fat1 promotes n-3 polyunsaturated fatty acid synthesis and expression of genes involved in lipid metabolism in goat cells.

The n-3 fatty acid desaturase gene fat1 codes for the n-3 desaturase enzyme, which can convert n-6 polyunsaturated fatty acids (PUFAs) to n-3 PUFAs. The n-3 PUFAs are essential components required for normal cellular function and have preventive and therapeutic effects on many diseases. Goat is an important domestic animal for human consumption of meat and milk. To elevate the concentrations of n-3 PUFAs and examine the regulatory mechanism of fat1 in PUFA metabolism in goat cells, we successfully constructed a humanized fat1 expression vector and confirmed the efficient expression of fat1 in goat ear skin-derived fibroblast cells (GEFCs) by qRT-PCR and Western blot analysis. Fatty acid analysis showed that fat1 overexpression significantly increased the levels of total n-3 PUFAs and decreased the levels of total n-6 PUFAs in GEFCs. In addition, qRT-PCR results indicate that the FADS1 and FADS2 desaturase genes, ELOV2 and ELOV5 elongase genes, ACO and CPT1 oxidation genes, and PPARa and PPARγ transcription factors are up-regulated, and transcription factors of SREBP-1c gene are down-regulated in the fat1 transgenic goat cells. Overall, fat1-overexpression resulted in an increase in the n-3 fatty acids and altered expression of PUFA synthesis related genes in GEFCs. This work lays a foundation for both the production of fat1 transgenic goats and further study of the mechanism of fat1 function in the PUFAs metabolism.

Scd1 mammary-specific vector constructed and overexpressed in goat fibroblast cells resulting in an increase of palmitoleic acid and oleic acid.

Stearoyl-CoA desaturase-1 (Scd1) is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Overexpression of Scd1 in transgenic animals would modify the nutritional value of ruminant-derived foods by increasing the monounsaturated fatty acid (MUFA) and decreasing the saturated fatty acid (SFA) content. The aim of this study was to develop an effective Scd1 vector that is specifically expressed in dairy goat mammary glands. We successfully amplified the goat full length Scd1 cDNA and evaluated its activity in goat ear skin-derived fibroblast cells (GEFCs) by lipid analysis. In addition, we constructed a mammary gland-specific expression vector and confirmed efficient expression of Scd1 in goat mammary epithelial cells (GMECs) by qRT-PCR and Western blot analysis. Fatty acid analysis showed that Scd1-overexpression resulted in an increase in levels of palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), from 1.73 ± 0.02% to 2.54 ± 0.02% and from 27.25 ± 0.13% to 30.37 ± 0.04%, respectively (both p<0.01) and the ratio of MUFA to SFA was increased. This work lays a foundation for the generation of Scd1 transgenic goats.

Growth regulation, imprinting, and epigenetic transcription-related gene expression differs in lung of deceased transgenic cloned and normal goats.

Somatic cell nuclear transfer (SCNT) is a promising technique to produce mammalian transgenic clones. Only a small proportion of manipulated embryos, however, can develop into viable offspring. The abnormal growth and development of cloned animals, furthermore, are accompanied by aberrant lung development. Our objective was to investigate molecular background of lung developmental problems in transgenic (random insertion of exogenous DNA) cloned goats. We examined expression of 15 genes involved in growth regulation, imprinting, and epigenetic transcription in lung tissue of deceased transgenic cloned and normal goats of various ages. Compared with normal goats of the same age from conventional reproduction, expression of 13 genes (BMP4, FGF10, GHR, HGFR, PDGFR, RABP, VEGF, H19, CDKNIC, PCAF, MeCP2, HDAC1, and Dnmt3b) decreased in transgenic cloned goats that died at or shortly after birth; Expression of eight genes (FGF10, PDGFR, RABP, VEGF, PCAF, HDAC1, MeCP2, and Dnmt3b) decreased in fetal death of transgenic cloned goats. Expression of two epigenetic transcription genes (PCAF and Dnmt3b) decreased in disease death of transgenic cloned goats (1-4 months old). Disruptions in gene expression might be associated with the high neonatal mortality in transgenic cloned animals. These findings have implications in understanding the low efficiency of transgenic cloning.

Age-associated changes in gene expression of goat oocytes.

Oocyte aging severely decreases the quality of oocytes, which hampers fertilization and subsequent embryo development. In the present study, age-dependent molecular changes in goat oocytes were investigated. First, the quality of goat oocytes with various in vitro culture times (24, 30, 36, 48, and 60 hours) was evaluated on the basis of developmental rates of parthenogenetically activated embryos and apoptosis of cumulus cells (CCs). Second, relative gene expression of six genes (mitochondrial genes: PGC-1α and NRF-1; epigenetic modification genes: SNRPN and HAT1; mitotic spindle checkpoint protein: SMAD2; and hyaluronan synthase gene: HAS3) were analyzed during oocyte aging. Third, we further studied the changes of seven genes (PGC-1α and NRF-1; apoptotic-related genes: BAX and BCL2; hyaluronan synthase gene: HAS2; metabolism-related gene: STAR; and superoxide dismutase gene: SOD1) in CCs during oocyte aging. In these studies, the blastocyst rate gradually decreased and the number of apoptotic cells significantly increased as the culture time increased (P < 0.05). Moreover, relative gene expressions of PGC-1α, NRF-1 and SMAD2 significantly decreased from 24 to 36 hours (P < 0.05), whereas the levels of HAT1 and HAS3 slowly increased as culture was prolonged. Furthermore, the levels of PGC-1α, BCL2, HAS2 and SOD1 quickly reduced, and BAX significantly increased from 24 to 36 hours in aged CCs (P < 0.05). In conclusion, goat oocytes started to age at 30 hours in vitro culture, and gene expression patterns of oocytes and CCs significantly changed as the oocytes aged. Gene expression pattern changes in CCs may provide a convenient and effective way to detect oocyte aging without compromising oocyte integrity.

Production of myostatin-targeted goat by nuclear transfer from cultured adult somatic cells.

Myostatin, a member of the transforming growth factor-ß family, acts as a negative regulator of skeletal muscle mass. In this study, myostatin-targeted caprine fibroblasts were obtained and subjected to SCNT to determine whether myostatin-knockout goats could be created. Fibroblasts from a 2-mo-old goat were transfected with a myostatin-targeted vector to prepare transgenic donor cells for nuclear transfer. After serum-starvation (for synchronization of the cell cycle), the percentage of transgenic fibroblasts in the G(0)/G(1) phase increased (66.2% vs. 82.9%; P < 0.05) compared with that in the control group, whereas the apoptosis rate and mitochondrial membrane potential were unaffected (P > 0.05). There were no significant differences between in vivo- and in vitro-matured oocytes as recipient cytoplasts for rates of fusion (86.5% vs. 78.4%), pregnancy (21.6% vs. 16.7%), or kidding (2.7% vs. 0%). One female kid from an in vivo-matured oocyte was born, but died a few hours later. Microsatellite analysis and polymerase chain reaction identification confirmed that this kid was genetically identical to the donor cells. Based on Western blot analysis, myostatin of the cloned kid was not expressed compared with that of nontransgenic kids. In conclusion, SCNT using myostatin-targeted 2-mo-old goat fibroblasts as donors has potential as a method for producing myostatin-targeted goats.

Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin.

The objective was to investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene nucleus transfer dairy goats. Three transfected cell lines from ear biopsies from three 3-mo-old Saanen dairy goats (designated Number 1, Number 2, and Number 3, respectively) were selected as karyoplast donors for somatic cell nuclear transfer (SCNT) after detailed identification (including PCR and sequencing of PCR products). In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 days of serum starvation or 2 days of contact inhibition. Additionally, there was no effect (P > 0.05) on developmental capacity of reconstructed embryos; however, the kidding rate of recipients in the serum starvation group was higher than that in the contact inhibition group (18 vs. 0%, respectively). The production efficiency of the transgenic cloned goats using donor cells from the Number 1 dairy goat cell line was higher than those using the Number 2 and the Number 3 cell lines (kidding rates were 18, 2, and 0%, respectively, P < 0.05). The oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-mo-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT.

Effect of different levels of short-term feed intake on folliculogenesis and follicular fluid and plasma concentrations of lactate dehydrogenase, glucose, and hormones in Hu sheep during the luteal phase.

This study investigated the effects of short-term food restriction or supplementation on folliculogenesis and plasma and intrafollicular metabolite and hormone concentrations. Ewes were randomly assigned to three groups: the control group received a maintenance diet (M) while the supplemented group and restricted group received 1.5×M and 0.5×M respectively on days 6-12 of their estrous cycle. Estrus was synchronized by intravaginal progestogen sponges for 12 days. On days 7-12, blood samples were taken. After slaughter, the ovarian follicles were classified and the follicular fluid was collected. Compared with restriction, supplementation shortened the estrous cycle length, decreased the number of follicles 2.5-3.5 mm and follicular fluid estradiol (E2) concentration, increased the number of follicles>3.5 mm and plasma glucose, insulin and glucagon concentrations, and augmented the volume of follicles>2.5 mm. Restricted ewes had higher intrafollicular insulin concentration, but it was similar to that of supplemented ewes. Compared with follicles≤2.5 mm, the intrafollicular glucose and E2 concentrations were increased and the testosterone, insulin, and glucagon concentrations and lactate dehydrogenase (LDH) activity were decreased in follicles>2.5 mm. Only in restricted ewes were intrafollicular LDH and testosterone concentrations in follicles≤2.5 mm not different from those in follicles≤2.5 mm. In conclusion, the mechanism by which short-term dietary restriction inhibits folliculogenesis may involve responses to intrafollicular increased E2, testosterone, and LDH levels in late-stage follicles. This may not be due to the variation of intrafollicular insulin level but rather due to decreased circulating levels of glucose, insulin, and glucagon.