Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.     

Nutritional evaluation of organically grown fodders in lactating Murrah buffaloes (Bubalus bubalis)

A study was undertaken to evaluate the nutritional response of organically grown diets in buffaloes on nutrients utilization and nutrient efficiency for milk production. For this, ten milch Murrah buffaloes of average body weight (490.72?±?6.65 kg), milk yield (8.13?±?0.33 kg), and lactation stage (85?±?5.28 days) were distributed in inorganic (InDg₎ and organic dietary groups (OrDg) with five animals in each. Buffaloes of InDg and OgDg were fed sorghum hay–berseem fodder-concentrate mixture-based diets grown inorganically and organically, respectively. After 60 days of feeding, a digestion cum metabolism trial was conducted to assess feed intake, nutrient utilization, and N balance, while milk yield and composition were recorded fortnightly. DM, CP, digestible crude protein (DCP), and metabolizable energy (ME) intake (g/Kg w^0.75) were similar in animals of InDg and OrDg. NDF, ADF, and hemicellulose digestibility were (P?<?0.05) higher in animals on OrDg (59.20, 51.55, and 62.67) than InDg diet (54.57, 43.72, and 56.61 %), respectively. Urinary n loss (g/day) was (P?<?0.05) lower in OrDg (67.23) than in InDg (83.55); however, milk N was comparable in animals of both dietary groups (47.36 vs 45.82 g/day). Nitrogen balance was higher in animals of OrDg (39.72) than in InDg (28.08). DCP, TDN, and ME values of both diets were similar. No effect of diet was observed on milk yield and its composition; however, increased lactation length decreased milk yield. Buffaloes on both diets had similar efficiency and conversion ratio of nutrients for milk production. Results revealed that diets (organically vs. inorganically grown) have no effect on milk yield and its composition; however, buffaloes on organic diet have higher fiber digestibility and low urinary N loss which did not affect the dietary nutrients efficiency and conversion ratio for milk production.     

定时输精对奶牛第一情期受胎率的效果分析

[目的]探讨不同月份和精液类型对利用定时输精程序处理奶牛后第一情期受胎率的影响.[方法]选择93头产后60 d以上不发情和产后配1～3次仍未受胎的奶牛,不检查卵巢直接利用激素生源2+1进行同期发情处理.1～3月份处理41头奶牛,6～8月份处理52头;其中,利用性控精液配种58头,常规精液配种35头.[结果]表明:经定时...     

Factors Affecting the Quality of Cryopreserved Buffalo (Bubalus bubalis) Bull Spermatozoa

Storage of buffalo ( Bubalus bubalis ) bull semen in the cryopreserved state is discussed in this article. Fertility rate in buffalo following artificial insemination with frozen–thawed semen is reviewed. To better understand the freezability of bubaline spermatozoa, the available data on biochemical components and the activity of specific enzymes of semen/spermatozoa are given. Moreover, the major factors that may influence the post-thaw viability and fertility of buffalo spermatozoa are examined in detail. In addition, suggestions for improvement in cryogenic procedures for buffalo spermatozoa are also given.     

Light and Scanning Electron Microscopic Studies of Oviductal Epithelium in Thai Swamp Buffalo (Bubalus bubalis) at the Follicular and Luteal Phases

The purpose of this study was to investigate the morphological changes in the epithelium of Thai swamp buffalo oviducts at the follicular and luteal phases by histological technique and scanning electron microscopy. The samples from the infundibulum, ampulla, isthmus and uterotubal junction (UTJ) of the oviduct were taken immediately after slaughter at the local abattoir. Noticeable cyclic changes were observed on the epithelial surface of the infundibulum and ampulla, but few changes were present in the isthmus and UTJ. At the follicular phase, the epithelium of infundibulum and ampulla were densely covered with ciliated cells whose cilia concealed the apical processes of the secretory cells. In contrast, the secretory cells dominated in the epithelium at the luteal phase and most of the ciliated cells were hidden by the bulbous processes of these cells. In the isthmus and UTJ at the follicular and luteal phases, the secretory cells were almost flat or gently rounded and covered with numerous microvilli at their apical surface. In conclusion, the histological and ultrastructural observation of Thai swamp oviduct epithelium revealed marked cyclic changes in the cellular differences associated with the main functions of segmental variations.     

Evidence of Lamina muscularis mucosae in Rumen of Buffalo (Bubalus bubalis)

Histological sections were studied from 4 sites of the rumen of 22 buffaloes, aged from 1 day to over 18 years of age. The sections were stained with Masson's trichrome stain. A definite layer of smooth muscle cells, representing the lamina muscularis mucosae separating the propria from the submucosa and extending into the ruminai papillae, was observed in buffaloes over 1.5 years of age. In animals over 10 years, the smooth muscle cells were very thin and elongated.     

Molecular Characterization of Oxytocin Receptor Gene in Water Buffalo (Bubalus bubalis)

Buffaloes are known for their productivity as compared to average yielding cows due to higher fat percentage, better feed conversion ability and disease resistance. On the other hand, the reproductive performances of buffaloes are often considered as poor owing to late sexual maturity, weak/silent oestrus, repeat breeder and prolonged intercalving interval. The study of cascade of events during oestrus and oestrous cycle can be useful for the improvement of reproductive efficiency of buffaloes. More precisely, the hormonal changes initiated at the molecular level within the animal determine the reproductive nature of the species. Nucleotide/protein sequence analysis serves as a vital tool in analysing the binding of the hormones for their effect or functions. In this study, we have reported cloning and characterization of the complete coding (cDNA) sequence of oxytocin receptor gene (OXTR) in buffaloes. Buffalo OXTR gene contains an uninterrupted ORF of 1176 nucleotides corresponding to an inferred polypeptide length of 391 amino acids (aa). The molecular weight of the deduced aa sequence was found to be 43 kDa with an isoelectric point of 9.253 and 16.328 charge at pH 7.0. The deduced protein sequence consists of 38 strongly basic (+) (K,R), 22 strongly acidic (?) (D,E), 186 hydrophobic (A, I, L, F, W, V) and 95 Polar (N, C, Q, S, T, Y) aa. Results indicated that aspartate (D) at aa position 85 and D, R and C at aa positions 136, 137 and 138, respectively, are conserved in buffaloes. The buffalo OXTR gene shared a per cent similarity ranging from 84.7 to 98.1 and 88.5 to 97.7 at nucleotide and deduced aa sequence levels, respectively, with that of other species. Phylogram constructed on the basis of either nucleotide or deduced aa sequences of buffalo OXTR gene showed that buffalo, cattle and sheep have diverged from human and swine and formed a separate clad. The buffalo sequence has shown maximum similarity and closeness with cattle followed by sheep both at nucleotide and at aa level.     

中国水牛遗传育种研究进展

我国水牛遗传育种的研究主要集中在染色体核型、蛋白质多态性、水牛胚胎体外生产与胚胎移植技术等方面。本文从中国水牛的类型和分布特征、遗传特性的研究现状、现代生物技术在水牛育种中的应用、中国水牛育种研究前景及展望等四个方面综述水牛的育种进展,认为以现代生物技术为核心的分子育种将成为水牛育种的总趋势,为我国水牛的产业化发展提供了理论依据。     

An Ultrastructural Study of the Sertoli Cell in the Water Buffalo (Bubalus bubalis)

The ultrastructure of Sertoli cell in the water buffalo (Bubalus bubalis) was observed in a transmission electron microscope. The nucleus had homogeneous nucleoplasm, scarce heterochromatin and multivesicular nuclear body (MNB). The MNB was composed of numerous vesicles and ribosome-like dense structures. The vesicles varied in size and number and contained a sparse and flocculent substance. In the indentation of the nucleus, aggregates of ribosomes were frequently observed. In the apical and middle region of the cell, long mitochondria and microtubules were distributed parallel to the long axis of the cell. Non-laminated smooth ER and some ribosomes were also recognizable throughout this region. In the basal region, widely-distributed laminated smooth ER was characteristic. Microfilament bundles at ectoplasmic specialization were irregularly arranged. Frequently-emerged nodular processes occasionally separated from basal lamina and formed round structures within Sertoli cytoplasm. Although these characteristics of buffalo Sertoli cell were very similar to those of the bovine studied, the aggregate of ribosomes was more developed in the buffalo.     

水牛κ-酪蛋白基因（CSN3）外显子4序列多态性研究

 分别设计位于CSN3 5个外显子旁侧引物,采用DNA测序法对沼泽型和河流型水牛CSN3编码区结构进行了分析,并对10个水牛群体共106个样本CSN3第4外显子序列进行了变异检测。结果表明,两类水牛CSN3编码区由848个核苷酸组成,包括ORF序列573 bp、5′-UTR序列69 bp和3′-UTR序列206 bp。在水牛CSN3第4外显子中共检测到4个SNP,其中c.445G>A,c.467C>T和c.516A>C为异义替换,导致相应的κ-CN成熟肽中氨基酸发生p.Val128Ile、p.Thr135Ile和p.Glu151Asp改变,c.467C>T和c.516A>C替换可能对κ-CN功能产生了影响。群体遗传分析表明,在河流型水牛中,等位基因c.445G、c.467C、c.471C和c.516A均为优势等位基因,而在沼泽型水牛中,仅c.445G、c.467C和c.471C为优势等位基因,河流型和沼泽型水牛的遗传差异主要体现在SNP516位点的群体遗传组成上。     

Production of Buffalo (Bubalus bubalis) Embryos in vitro: Premises and Promises

Techniques for in vitro production (IVP) of buffalo embryos adopting the procedures developed in cattle have received increasing interest in the recent times. A high oocyte maturation, fertilization and cleavage rate and a low rate of blastocyst yield and calving following transfer of in vitro produced buffalo embryos have been obtained. The efficiency of IVP in buffalo is much lower than that in cattle. Several problems need to be resolved before IVP technology can be used regularly in buffalo breeding. This review attempts to present an overview of the different techniques used in buffalo to produce transferable embryos in vitro, namely in vitro maturation and fertilization of immature oocytes and in vitro development of the resulting cleaved embryos to the blastocyst stage before transfer. The problems associated with IVP, the possible solutions and the new biotechniques linked to IVP are discussed.     

The Epididymis of the Prepubertal Swamp Buffalo (Bubalus bubalis): Histochemistry of Phosphatases

The distribution of phosphatases was examined in the epididymis of swamp buffaloes aged 1.5-2 years, where spermatogenic activity in the testis had reached the early primary spermatocyte stage. A granular distribution of acid phosphatase was present in the luminal region of the epithelium of the efferent ductules. In the ductus epididymidis, four zones were identified and all zones showed varying degrees of acid-phosphatase activity in the epithelium, with the most pronounced activity in the apical region and stereocilia of zone II. Alkaline-phosphatase activity occurred along the basal region of the epithelium in zones I. III and IV of the ductus and in the stereocilia of zones I and II. An intense apical reaction was seen in zone II. The efferent ducts were free of this enzyme. Thus, zone II is considered the most active region, having the function of absorption and possibly steroid metabolism.     

Transplacental Transfer of Iron in the Water Buffalo (Bubalus bubalis): Uteroferrin and Erythrophagocytosis

The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental‐maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl’s reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal‐placental tissues in buffalo throughout pregnancy.     

Anatomical Studies of the Accessory Nerve in the Indian Buffalo (Bubalus bubalis)*

This is a study of the origin, formation, course, distribution, the fascicular number, their area and arrangement, total fibre count, density and diameterspectra of the accessory nerve in the Indian buffalo. The radices spinales are located from the level of C₁ to midlevel of C₆cord segments. The radices craniales arise in series from the medulla oblongata caudal to those of the vagus nerve. The accessory nerve contains 7931 myelinated fibres arranged in 31 fascicles. Of these 41 % have an external diameter of 10–12 μm. The greatest density of fibres viz. 1781/mm² is obtained in the anastomosing ramus from the second cervical spinal nerve. The least value (377/mm²) is found in its ventral ramus. However, the dorsal ramus appears to have a larger cross-sectional area.     

Milk Progesterone Profile at and after Artificial Insemination in Repeat-Breeding Cows: Effects on Conception Rate and Embryonic Death

The aim of this study was to investigate whether the skim milk progesterone concentrations at artificial insemination (AI) and day of rise of post-ovulatory progesterone concentration thereafter affect the conception and embryonic death rates in repeat-breeding cows. Milk samples were obtained from 96 repeat-breeding cows that failed to conceive to three or more AIs. The samples were taken from the cows at the day of AI and three times/week until day 45 post-AI. Skim milk was obtained after centrifugation and used for progesterone assay. The cows with a progesterone concentration more than 0.5 ng/ml at AI showed a significantly higher incidence of late embryonic death than those having a progesterone concentration<0.5 ng/ml at AI (p<0.01). As the progesterone level at insemination rose, conception rate declined. A negative correlation was shown between conception rate and skim milk progesterone level at AI. Of 56 cows showing a rise of progesterone to 1 ng/ml or more within 6 days after AI, 28 cows (50%) conceived. On the contrary, only eight of 39 cows (20.5%) conceived when the progesterone rose up to 1 ng/ml after day 6 post-AI. We concluded that increased progesterone concentration at the time of AI and delayed rise of progesterone post-AI might lead to decrease in fertility in repeat-breeding cows.