Animal Biotechnology Roles in Livestock Production

Currently, meat and milk productions are significantly increasing especially in Asia. The supply of these products is vital to people's health and well-being, whereas the efficiency of beef production appears to be still lower than other meat productions. Improvements in the quality and functionality of their livestock products, as well as their production efficiency, are required for further production. Animal biotechnologies have contributed to genetic improvement, genetic diversity maintenance of domestic animals, etc. Basic animal biotechnologies, such as artificial insemination and embryo transfer, have been well established and applied as powerful tools for genetic improvement of livestock. In the applications of artificial insemination techniques, the use of sexed semen has been now widely spread, and also efforts are also made in the development of the technology using a small amount of sperm. For embryo transfer, several types of vitrification technologies have been applied to improve pregnancy rates and contributed to the international/domestic supply of livestock embryos. Conventional animal biotechnologies, such as in vitro fertilization and intracellular sperm injection, have been applied to not only livestock production and also human-assisted reproductive medicine. For in-vitro production of embryos in domestic animals, currently, oocytes have been collected from medium or large follicles (3-6 mm or larger in diameter) of ovaries. Although the oocytes derived from small follicles (less than 3 mm in diameter) exist more on the surface of ovaries, the developmental competence of the oocytes has been known to be significantly lower than those from medium follicles. If we could improve the competence of oocytes derived from small follicles significantly, we may be able to increase the number of female gamete resources for in vitro embryo production. Also, the development of techniques for producing transgenic and cloned animals has greatly contributed to the creation of pharmaceuticals and organs for xenotransplantation. Recently, furthermore, genome editing technologies, such as combined use of CRISPR/Cas9 and PiggyBac, have been developed and have made it possible to correct specific parts of the genome and introduce mutations by homologous recombination. In this review, I would like to discuss the application and progress of the above biotechnologies, including our recent research results

前核形成および初期胚発生に関係する豚卵胞卵子の細胞質成熟

Successful in vitro production of production of porcine embryos requires a series of integrated, effective techniques for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). This paper reviews about cytoplasmic maturation associated with the efficiency of in vitro production of porcine embryos. Traditionally, the failure to from a male pronucleus have been reported as serious problems in producing porcine emabryos following IVM and IVF. The problem of male pronuclear formation is currently considered to be mainly due to oxidative stress during IVM. More recently the developmental competence of embryos following IVM and IVF has been investigated through improvement of culture conditions for oocyte maturation. Currently, an acceptable rate of blastocyst formation and the birth of live piglets has been achieved by investigating affecting factors during IVM, IVF and IVC of porcine oocytes. Since the ovarian oocytes available for IVM are primarily those present in mid-size antral follicles of prepuberal gilts, more research is needed to gain an improved understanding of the factors associated with the developmental competence in oocytes from both preantral and antral follicles.豚受精卵の体外生産を成功させるためには、体外成熟、体外受精および対外発生に必要な効率的な一連の技術の積み重ねが必要である。本総説は、効率的な豚受精卵の体外生産に関係する体外成熟中の諸要因について論じる。長い間、体外成熟卵子の体外受精後の雄性前核の形成不全は、体外受精卵を生産するための深刻な問題として、多くの研究者によって長い間研究されてきた。しかし現在では、雄性前核形成に関するこの問題は、すでに解決され、主に体外成熟中の酸化ストレスが原因であると考えられている。さらに最近、体外成熟・受精卵の初期発生能力は、卵成熟期間中の培養条件の改善を通じて飛躍的に改善されており、現在では、豚受精卵の初期発生に影響をおよぼす卵子成熟中の諸要因について研究によって、産業的に利用可能な胚盤胞受精卵作用効率およ産仔の生産効率が得られている。体外成熟可能な卵胞卵子は、通常春機発動以前の屠畜雌豚の卵巣に存在する中型成熟卵胞から回収されている。そこで、今後の研究において、春機発動以前の雌豚および性成熟を経た雌豚のそれぞれに存在する卵胞卵子の発生能力に影響をおぼす要因のさらなる理解が必要とされる

Effect of glucose and pyruvate on nuclear and cytoplasmic maturation of porcine oocytes in a chemically defined medium

The objective was to examine potential roles of glucose and pyruvate in nuclear and cytoplasmic maturation of porcine oocytes. Oocyte-cumulus complexes (OCC), derived from 3 to 6 mm follicles, were cultured in a chemically defined medium (pyruvate-free mNCSU37-PVA), with or without 5.55 mM glucose, during in vitro maturation (IVM); in the absence of glucose, germinal vesicle breakdown (GVBD) and nuclear maturation were prevented (P &#60; 0.05). Subsequently, OCC were cultured for IVM in glucose-containing mNCSU37-PVA supplemented with 6-amononicotinamide (6-AN) and diphenyleneiodonium (DPI), inhibitors of the pentose phosphate pathway (PPP); both compounds (&#62;= 10 mu M 6-AN and &#62;= 10 nM DPI) inhibited resumption of meiosis (P &#60; 0.05). Supplementation of glucose-free maturation medium with increasing concentrations of pyruvate induced resumption of meiosis and increased the incidence of oocytes reaching metaphase-II in a concentration-dependent manner (P &#60; 0.05). More mature oocytes were obtained in the presence of pyruvate + glucose (P &#60; 0.05). After culture to allow maturation, glutathione content was higher in oocytes cultured in the presence of pyruvate alone than in those cultured in glucose alone; inclusion of 6-AN abolished responses to pyruvate (P &#60; 0.05). In conclusion, both glucose and pyruvate played a critical role in the release of porcine oocytes from arrest at the GV-I stage, probably through the PPP, whereas supplementation with pyruvate improved cytoplasmic maturation, as determined by oocyte glutathione content.</p

In-vitro Culture with a Tilting Device in Chemically Defined Media During Meiotic Maturation and Early Development Improves the Quality of Blastocysts Derived from In-vitro Matured and Fertilized Porcine Oocytes

Under physiological conditions, mammalian oocytes and embryos appear to be stimulated not only chemically but also mechanically, such as by compression, shear stress and/or friction force in the follicle and female reproductive tract. The present study was undertaken to examine the effects of kinetic culture with a tilting device in chemically defined media during in vitro maturation (IVM) of porcine oocytes and in vitro culture (IVC) following in vitro fertilization (IVF) on the early developmental competence and quality of blastocysts. After culture in a chemically defined IVM medium, modified porcine oocyte medium (mPOM) containing gonadotropins and dibutyryl cAMP for 20 h, the mean diameter of the cumulus-oocyte complexes (COCs) was larger in the tilting culture than in the static controls, whereas the diameter of the oocytes did not differ. When culture of the COCs was continued additionally in a fresh medium without gonadotropins and dibutyryl cAMP for 24 h, the incidences of oocytes completing GVBD and developing to the metaphase-II stage did not differ between the tilting and static culture systems. Furthermore, the sperm penetration after IVF and developmental competence of the oocytes to the blastocyst stage were not different between the tilting and static systems during IVM and IVC. However, tilting culture during both IVM and IVC had a significant positive effect on the number of cells per blastocyst (P<0.05). These observations indicate that tilting culture during IVM and IVC in chemically defined media improves the quality of blastocyst, as determined by the number of cells per blastocyst, without any effects on penetrability and developmental competence

Hydrophobic Silicone Elastomer Chamber for Recording Trajectories of Motile Porcine Sperms without Adsorption

Motile porcine sperms adhere to hydrophilic materials such as glass and plastics. The adsorption of sperms to a hydrophobic poly(dimethylsiloxane) (PDMS) membrane is less compared with that to glass. We investigated the linear velocity (LV) and amplitude of lateral head displacement (ALHD) of motile porcine sperm on glass and PDMS preparations using computer-assisted sperm analysis (CASA). Significant decreases were observed in the 15-min LV (P<0.05) and ALHD (P<0.05) in motile porcine sperm on glass preparations compared with those on PDMS preparations. These differences were due to adsorption of the head and/or neck to hydrophilic substrates. Because of the elasticity of PDMS, we propose that a PDMS membrane should be used for CASA. To investigate the dynamics of motile porcine sperms with microfluidics, we do not recommend plasma treatment to bond PDMS and glass in the microchannel preparation; instead, we suggest that a PDMS molding process without plasma treatment be used for preparation of microfluidic channels

Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration

The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 x 10(8) with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 +/- 0.5 x 10(4) to 575.0 +/- 56.3 x 10(4) cells/mL; mean +/- SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 +/- 10.5 to 100%), but the monospermic penetration rate was lower (P < 0.05) in oocytes 3.5 mm from the exit position (12.5 +/- 4.8%) than those at 7.5 mm (53.1 +/- 6.0%) or further (41.9 +/- 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P < 0.05) in the MFSS-IVF system (0.375 +/- 0.040) than both standard IVF and transient IVF (0.222 +/- 0.028 and 0.189 +/- 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P < 0.05) in the MFSS-IVF system (40.9 +/- 2.3%) than in either standard or transient IVF (22.6 +/- 1.4 and 33.7 +/- 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber

Effect of Oral Administration of Dehydroepiandrosterone on PCOS-Like Phenotype of Female C57BL/6 Mice

We aim to evaluate the efficacy and optimal dose of orally administered DHEA in the PCOS mice model by assessing their ovarian morphology and serum FSH, LH, and testosterone levels. Female C57BL/6 mice were divided into a control group (n=5, received daily injections of 0.2 ml sesame oil) and an experimental group, which was further classified into 1) D-45 group (n=5), 2) D-60 (n=5), and 3) D-90 group (n=5) that were treated with 45, 60, and 90 mg/Kg body weight of oral DHEA. After modelling, mice in the control group had a regular estrous cycle, while mice in all experimental groups had a disturbed estrous cycle. Ovarian histology showed several growing follicles and some corpora lutea (CL) in the control, D-60, and D-90 groups. However, some large antral follicles and decreased CL were observed in the D-45 group. Serum FSH was significantly lower in the D-45 group than in the control group (3.73 ± 0.12 vs. 5.28 ± 0.31 mIU/mL, P&lt;0.01), but D-60 and D-90 groups had a similar FSH level to the control group (P&gt;0.05). The serum level of LH and testosterone were significantly elevated in the D-45 group than in the control group (2.52 ± 0.43 vs. 1.30 ± 0.33 mIU/mL, P&lt;0.01 and 1.80 ± 0.32 vs. 1.24 ± 0.23 ng/mL, P&lt;0.01, respectively). Still, the level of LH and testosterone in the D-60 and the D-90 groups was comparable to the control group (P&gt;0.05). Our study suggests that oral administration of DHEA is efficacious in establishing PCOS-like phenotype in mice with the suggested optimal dosage of 45 mg/Kg body weight