Localization of leptin and leptin receptor in the bovine adenohypophysis

The present study was carried out to detail the cellular localization of leptin (Lep) and the leptin receptor (LepR) in the bovine adenohypophysis. Lep immunoreactivity (Lep-ir) was found in about 30% of adenohypophysial cells in the gland. Immunochemistry of Lep and specific hormones using serial sections revealed that Lep-ir was present in 60.4% of somatotrophs, 15.9% of gonadotrophs, 6.5% of mammotrophs, 6.5% of thyrotrophs and 2.4% of corticotrophs. Both the common short isoform (OBRa) and the long isoform (OBRb) of LepR mRNA were expressed in the bovine adenohypophysis. LepR immunoreactivity (LepR-ir) was found in only 2.8% of the adenohypophysial cells and over 50% of LepR-ir cells were gonadotrophs, in which most of the cells were distributed in the zona tuberalis. The findings on Lep and LepR in the adenohypophysial cells indicate that Lep may regulate gonadotroph function through autocrine/paracrine pathway in the bovine adenohypophysis.     

Conditioning of karyoplasts for producing somatic nuclear transferred gonadal germ cells in domestic chickens

The objective of this study was to establish a protocol for generating karyoplasts that can be used to produce somatic nuclear transferred gonadal germ cells (snt-GGCs) in domestic chickens. Karyoplasts were produced by centrifuging cultured fibroblasts from 10-day-old chick embryos at 10,000 x g in the presence of 1.0 microg/ml cytochalasin B. The number of karyoplasts was significantly (P<0.05) higher and the diameters of the karyoplasts were significantly (P<0.05) smaller when fibroblasts were centrifuged for 60 min than for 10 or 30 min. It was possible to generate snt-GGCs by electrofusion of GGCs with karyoplasts produced from cryopreserved or serum-starved fibroblasts. These results indicate that karyoplasts generated from 10-day-old chick embryos can be used to produce snt-GGCs even after cryopreservation and serum starvation of the fibroblasts.     

Health status and productive performance of somatic cell cloned cattle and their offspring produced in Japan

Since the first somatic cell cloned calves were born in Japan in 1998, more than 500 cloned cattle have been produced by somatic cell nuclear transfer and many studies concerning cloned cattle and their offspring have been conducted in this country. However, most of the results have been published in Japanese; thus, the data produced in this country is not well utilized by researchers throughout the world. This article reviews the 65 reports produced by Japanese researchers (62 written in Japanese and 3 written in English), which employed 171 clones and 32 offspring, and categorizes them according to the following 7 categories: (1) genetic similarities and muzzle prints, (2) hematology and clinical chemistry findings, (3) pathology, (4) growth performance, (5) reproductive performance, (6) meat production performance and (7) milk production performance. No remarkable differences in health status or reproductive performance were found among conventionally bred cattle, somatic cell cloned cattle surviving to adulthood and offspring of somatic cell cloned cattle. Similarities in growth performance and meat quality were observed between nuclear donor cattle and their clones. The growth curves of the offspring resembled those of their full siblings.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Selected Aspects of Advanced Porcine Reproductive Technology

In vitro fertilization (IVF) of in vitro matured (IVM) oocytes in pigs has become the most popular method of studying gametogenesis and embryogenesis in this species. Furthermore, because of recent advances in in vitro culture (IVC) of IVM–IVF embryos, in vitro production (IVP) of embryos now enables us to generate viable embryos as successfully as for in vivo -derived embryos and with less cost and in less time. These technologies contribute not only to developments in reproductive physiology and agriculture but also to the conservation of porcine genetic resources and the production of cloned or genetically modified pigs. However, in IVP, there still remains the problem of abnormal ploidy, which is caused by performing procedures under non-physiological conditions. In recent years, unique technologies such as intracytoplasmic sperm injection (ICSI) or xenografting of gonadal tissue into immunodeficient experimental animals have been developed to help conserve gamete resources. These technologies combined with IVP are expected to be useful for the conservation of gametes from important genetic resources. Here, we discuss the developmental ability and normality of porcine IVP embryos and also the utilization of ICSI and xenografting in advancing biotechnology in pigs.     

Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen‐thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real‐time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen‐thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24‐hr incubation post warming. Furthermore, immunostaining against double‐strand DNA revealed DNA damage in frozen‐thawed embryos. When frozen‐thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle‐treated embryos. In addition, cell‐free mtDNA content in medium was higher in case of resveratrol‐treated embryos than of vehicle‐treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen‐thawed embryos, mitochondria were removed during post‐thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.     

Length of lags in responses of milk yield and somatic cell score on test day to heat stress in Holsteins

We used daily records from provincial Japanese weather stations and monthly test‐day records of milk production to investigate the length of the lags in the responses of cows’ milk yield and somatic cell score (SCS) to heat stress (HS). We also investigated the HS thresholds in milk yield and SCS. Data were a total of 17,245,709 test‐day records for milk and SCS in Holstein cows that had calved for the first time between 2000 and 2015, along with weather records from 60 weather stations. Temperature–humidity index (THI) values were estimated by using average daily temperature and average daily relative humidity. Adjusted THI values were calculated by using temperature, relative humidity, wind speed, and solar radiation. The model contained herd, calving year, month of test day, age group, days in milk, and THI as a fixed effect. THIs for each day from 14 days before the test day until the test day were used to represent the HS effects. The HS occurring 3 days, and between 8 and 10 days, before the test day had the greatest effect on the milk yield and SCS, respectively. The threshold THI values for the HS effect were about 60–65 for both traits.     

Effects of preventing hyperphagia on glycolipid metabolic abnormalities in Spontaneously Diabetic Torii fatty rats

Spontaneously Diabetic Torii (SDT) fatty rats, made by introducing the fa allele of the Zucker fatty rat into the SDT rat genome, represent a new model of obese type 2 diabetes. SDT fatty ^fa/fa (SDT fatty) rats exhibit overt obesity, hyperglycemia, and hyperlipidemia from about six weeks of age, and this is associated with hyperphagia by an induced disorder of leptin action. The present study was conducted to elucidate whether suppression of hyperphagia can improve reduce abnormalities in SDT fatty rats. SDT fatty rats were subjected to pair-feeding with SDT fatty ^{+/ +} (SDT) rats from 6 to 26 weeks of age, and the effects on metabolic parameters and diabetic complications were assessed. Body weights of the pair-fed rats were similar with those of SDT rats during the experimental period. Improvement of hyperglycemia or hypertriglyceridemia was observed from 8 to 16 or 12 weeks of age in the pair-fed rats, but hypercholesterolemia was not entirely improved during the experimental period. We also examined mRNAs expression in liver, and found that the expression associated with glyconeogenesis, such as glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK), tended to decrease in the pair-fed rats, and the mRNA expression of hydroxymethylglutaryl-CoA (HMG-CoA) was elevated. Renal parameters, such as blood urea nitrogen and urinary albumin excretion, were improved in the pair-fed rats. The incidence or progression of diabetic complications, such as renal lesions and cataract, was reduced. In conclusion, suppression of hyperphagia in SDT fatty rats was effective in temporally improving hyperglycemia or hypertriglyceridemia, and reducing the incidence or progression of diabetic complications, but was ineffective in reducing hypercholesterolemia.     

The α2A‐adrenoceptor subtype plays a key role in the analgesic and sedative effects of xylazine

Xylazine, the classical α₂‐adrenoceptor (α₂‐AR) agonist, is still used as an analgesic and sedative in veterinary medicine, despite its low potency and affinity for α₂‐ARs. Previous pharmacological studies suggested that the α_2A‐AR subtype plays a role in mediating the clinical effects of xylazine; however, these studies were hampered by the poor subtype‐selectivity of the antagonists used and a lack of knowledge of their bioavailability in vivo. Here, we attempted to elucidate the role of the α_2A‐AR subtype in mediating the clinical effects of xylazine by comparing the analgesic and sedative effects of this drug in wild‐type mice with those in α_2A‐AR functional knockout mice using the hot‐plate and open field tests, respectively. Hippocampal noradrenaline turnover in both mice was also measured to evaluate the contribution of α_2A‐AR subtype to the inhibitory effect of xylazine on presynaptic noradrenaline release. In wild‐type mice, xylazine (10 or 30 mg/kg) increased the hot‐plate latency. Furthermore, xylazine (3 or 10 mg/kg) inhibited the open field locomotor activity and decreased hippocampal noradrenaline turnover. By contrast, all of these effects were abolished in α_2A‐AR functional knockout mice. These results indicate that the α_2A‐AR subtype is mainly responsible for the clinical effects of xylazine.