Buffalo Fetuses Research Articles

Histomorphological, immunohistochemical, and ultrastructural study on ontogeny of ileocaecal lymphoglandular complexes in prenatal and postnatal Indian buffalo: An innate mucosal immune barrier.

The present study was conducted on prenatal and postnatal development of lymphoglandular complexes (LGCs) in ileocaecal region of buffalo fetuses (n=15) ranging from 11.5cm curved crown rump length (CVRL) (80 days) to 100 cm CVRL (299 days) and neonatal buffalo calves (n=10). The fetuses were categorized into three groups based on their CVRL. LGC formation was not evident in ileocaecal junction up to 32 cm CVRL (145 days). At 35 cm CVRL (152 days), diffuse lymphocytes were scattered around the base of glands that encircled them. At 54 cm CVRL (195 days), lymphoid aggregates were present in submucosa around deep submucosal glands and formed primordia of LGCs in ileocaeccal orifice region. At 100 cm CVRL (299 days), these complexes were completely visible grossly. The distinguished lymphoid nodules in submucosa were invaded by submucosal extensions of overlying mucosal glands to form a large clear complex of glands and lymphoid nodules called as "Lymphoglandular complex" at this stage. It is the first report of prenatal development of LGCs in large intestine of buffaloes. Abundant CD3+ T cells were observed towards periphery of LGC. In neonates, these complexes were uniform, few with demarcation into dark and light zones that is, germinal center formation. Lymphocytes interspersed in lamina propria were mainly CD3+ T lymphocytes. In conclusion, the development of LGCs in ileocaecal region started prenatally in terms of all its cellular components into completely developed and immunocompetent to generate mucosal immunity.

First Report on Abortion Caused by Salmonella enterica subsp. enterica Serovar Enteritidis in Water Buffalo (Bubalus bubalis).

Salmonella enterica subsp. enterica Serovar Enteritidis is one of the major pathogens associated with enteric diseases in animals and humans. Thus, due to the importance of Salmonella spp. infections for animal production and public health, the aim of the present study was to describe the first detection of S. enteritidis in an aborted water buffalo fetus in southern Italy by characterizing the phylogroup profile and the antimicrobial susceptibility of the isolated pathogenic strains. The different clinical manifestations of salmonellosis in animals include diarrhea, abortion, pneumonia, septic arthritis, meningitis, and others, depending on the virulence of the serovars, infectious dose, and host immunity. This study reports the first case of abortion caused by Salmonella enterica subsp enterica serovar Enteritidis in water buffalo (Bubalus bubalis) in the Campania region, southern Italy. Complete necropsy was performed on the aborted water buffalo fetus under study, and samples and swabs from different organs were collected. Samples were processed by microbiological and molecular analyses to detect bacterial, viral, and protozoarian pathogens possibly responsible for abortion. Whole genome sequencing (WGS) was carried out to further characterize the isolated S. Enteritidis strain. Our findings highlight the crucial role of S. Enteritidis as a potential abortive agent in water buffalo and its presence should therefore be investigated in cases of bubaline abortion.

Involvement of herpesviruses in cases of abortion among water buffaloes in southern Italy.

A six-year study on water buffaloes from the Campania Region (Southern Italy) was conducted to evaluate the presence of bovine/bubaline herpesviruses in cases of abortion. A total of 244 buffalo foetuses were analysed by real-time PCR to detect the presence of: bovine alphaherpesvirus 1(BoHV-1), bubaline alphaherpesvirus 1 (BuHV-1), bovine alphaherpesvirus 2 (BuHV-2), and bovine gammaherpesvirus 4 (BoHV-4). The foetuses of 14 water buffaloes that showed abortions were positive for BuHV-1 (4 animals) and/or BoHV-4 (11 animals), with one of these cases showing co-infection with BuHV-1 and BoHV-4. This study reports the first identification of BoHV-4 in water buffaloes. Cases of abortion were analysed using both molecular and cultural assays for the presence of other pathogens. In nearly all the abortion cases positive for BoHV-4, the virus was identified as a co-infecting agent together with other microorganisms, whereas in two abortion cases, it was the only pathogen found.

Assessment of prenatal ovarian and serum estrogen concentration in Nili-Ravi buffalo fetus

Assessment of prenatal ovarian and serum estrogen concentration in Nili-Ravi buffalo fetus

Assessment of prenatal ovarian and serum estrogen concentration in Nili-Ravi buffalo fetus

Assessment of prenatal ovarian and serum estrogen concentration in Nili-Ravi buffalo fetus

Whole-Genome Sequencing-Based Characterization of a Listeria monocytogenes Strain from an Aborted Water Buffalo in Southern Italy.

Listeria monocytogenes is a Gram-positive pathogen causing life-threatening infections both in humans and animals. In livestock farms, it can persist for a long time and primarily causes uterine infections and encephalitis in farmed animals. Whole genome sequencing (WGS) is currently becoming the best method for molecular typing of this pathogen due to its high discriminatory power and efficiency of characterization. This study describes the WGS-based characterization of an L. monocytogenes strain from an aborted water buffalo fetus in southern Italy. The strain under study was classified as molecular serogroup IVb, phylogenetic lineage I, MLST sequence type 6, Clonal Complex 6, and cgMLST type CT3331, sublineage 6. Molecular analysis indicated the presence of 61 virulence genes and 4 antibiotic resistance genes. Phylogenetic analysis, including all the publicly available European L. monocytogenes serogroup IVb isolates, indicated that our strain clusterized with all the other CC6 strains and that different CCs were variably distributed within countries and isolation sources. This study contributes to the current understanding of the genetic diversity of L. monocytogenes from animal sources and highlights how the WGS strategy can provide insights into the pathogenic potential of this microorganism, acting as an important tool for epidemiological studies.

Developmental Changes in Sublingual Salivary Gland of Indian Buffalo during Prenatal and Neonatal Life

Background: The sublingual salivary gland being one of the major salivary glands contributes to a substantial amount of saliva secreted into the mouth. Saliva contains water, various enzymes, mucopolysaccharides and lubricating glycoproteins. It has an important role in providing lubrication for eating and vocalization, aid digestion and supply saliva for pH buffering. The study of prenatal development is prerequisite to understand the normal developmental biology of an organ. The documentation of normal foetal growth can serve as a guide for understanding the consequence of harmful influences at various stages of gestation. The present study was aimed at elucidating the histogenesis of sublingual gland of buffalo during prenatal as well as neonatal life.Methods: The study was carried out on 36 buffalo foetuses, during different stages of prenatal development, as well as 6 neonates. The fetuses were categorized into three groups based on their curved crown rump length (CVRL). The ontogenetic events in histogenesis of sublingual salivary gland of foetal and neonatal buffaloes were observed.Result: The primordial anlage of monostomatic and polystomatic parts of sublingual gland was observed at 39th day and 53rd day of development respectively. The primary ducts (interlobular) were first observed at 80th day of development. The lobulation of the gland was started at 107th day of development, whereas the capsule formation was initiated by the aggregation of mesenchymal tissue at 121st day of development. The duct system was completed at 121st day of development. At same day, the terminal tubules attained the structure of the acini. The typical compound tubulo-acinar nature was first observed at 122nd day of development. The gland showed predominantly mucous type of cells at 137th day of development. The myoepithelial cells were first appeared at 138th day of development. The monostomatic and polystomatic parts were clearly distinguishable by connective tissue at 170th day of development. In neonates, the gland was of compound tubulo-acinar nature with a well-defined capsule. Localization of acidic and neutral mucopolysaccharides was observed in mucous and goblet cells. Fine lipid droplets were observed in intralobular as well as interlobular connective tissue, however, phospholipids were found in the cell membrane of secretory acinar cells and ducts.

Histo-ontogenetic study of the parotid salivary gland of Indian buffalo.

The present study was aimed at elucidating the histogenesis of parotid gland of buffalo. The study was carried out on buffalo foetuses (n=36), during different stages of prenatal life. The foetuses were categorised into three groups based on their curved crown rump length (CVRL). The primordial anlage of parotid salivary gland was evident at 40th day of development whereas the primary ducts, in the form of cords, were first observed at 81st day of prenatal life. The capsule formation as well as the lobulation of the gland was initiated at 127th day. At 141st day, the duct system of gland was completed. The terminal tubules attained the structure of acini at 167th day. The myoepithelial cells first appeared as flattened basal cells initially around the developing acinar cells at 167th day. The typical compound tubulo-acinar nature of the gland was first observed at 185th day. Purely serous acinar cells were seen from 185th day onwards. The micrometrical studies revealed that the mean diameter of acinar cells, intercalated ducts, striated ducts and large ducts increased with the advancement of age. The serous acinar cells were devoid of acidic as well as neutral mucopolysaccharides in prenatal age groups; however, large ducts with goblet cells exhibited positive reaction. Combined PAS-AB method revealed mixed reaction in acinar cells as well as in large ducts. Fine lipid droplets were observed in intralobular as well as interlobular connective tissue; however, phospholipids were observed in the cell membrane of secretory cells and ducts.

Prenatal Development of Incisors in the Egyptian Buffalo (Bos bubalis)

Objective: The teeth play important roles in food mastication, prehension and defense against predators. Although several studies demonstrated the development of teeth in different mammalian species, no data are, to our knowledge, available in Egyptian buffalos. Therefore, the present investigation was conducted to study the development of incisor teeth in buffaloe. Design: Descriptive study. Animals: seventeen buffalo embryos and fetuses of both sexes were used. Their crown vertebral rump lengths (CVRL) ranged from 2 to 46 cm (equivalent to 38 - 177 days old). Procedure: Embryos and fetuses were fixed in 10% neutral buffered formalin and decalcified by 14% EDTA solution for several weeks. The samples were dehydrated, cleared and embedded in paraffin wax using standard techniques. Sections were cut on Leitz microtome and mounted on uncoated slides. For general histological structure, a selection of slides was routinely stained with haematoxylin–eosin and examined by the light microscope. Result: The present study reported for the first time that the prenatal development of incisors in buffalo passes through three sequential stages: the bud, cap and bell stages. Although each stage was easily differentiated based on its form and its distinct developmental features, the three stages were overlapped with each other’s. Generally, the bud stage was observed in 11 cm CVRL buffalo fetuses, meanwhile the cap and bell stages were detected in 23 cm and 30 cm CVRL fetuses respectively. Conclusion and clinical relevance: These results can be used as inductive index for determination of the ages of buffalo embryos and fetuses.

Differentiation of pancreatic endocrine islets in buffalo fetus

Pancreas of 24 buffalo foetii collected from abattoir and veterinary clinics, GADVASU, Ludhiana were studied. The buffalo foetii were divided into three groups after measuring their CVRL, viz. Group I (CVRL between 0–20 cm), Group II (CVRL above 20 cm and up to 40 cm) and Group III (CVRL above 40 cm) and their approximate age was calculated. The tissues were processed and paraffin sections were cut and stained with different histological stains. In Group I, at 7.5 cm CVRL (62 days), the endocrine cells were scattered either in between the developing acinar cells and primitive tubules or were localized within primitive tubules. The alpha cells were large, round or oval in shape having eosinophilic cytoplasm and large nucleus whereas beta cells were small basophilic cells. At 12.8 cm CVRL (86 days), small groups of cells were present containing purely alpha cells or purely beta cells. Well developed islets of Langerhans with ill-defined capsule were observed at 19 cm CVRL (114 days). At 10.7 cm CVRL (77 days), certain cells which were morphologically different from cells of islets of Langerhans and acinar cells started to assemble and many capillaries were invading these cells. At 12.8 cm CVRL (86 days), these assembled cells formed well developed islets called perilobular islets. These large sized islets were developing at the periphery of lobules. The perilobular islets underwent regressive changes whereas islet of Langerhans increased in number simultaneously.

Fetal Leydig cells in buffalo testis: Light and electron microscopic study

The present study was conducted on testis of 20 buffalo fetuses ranging from 2.5 cm to 20 cm curved crown rump length (CVRL). The tissues were processed for light and transmission electron microscopic study. The intertubular tissue was filled with primitive mesenchymal cells, developing Leydig cells and blood vessels. At 7.5 cm CVRL, some of the interstitial mesenchymal cells started developing into foetal Leydig cells and were androgen positive. These cells were characterized by their polygonal shape, eosinophilic cytoplasm and darkly stained spherical nucleus with prominent nucleoli at 9.5 cm CVRL. At 15.5 cm CVRL, the fetal Leydig cells were present in groups around the interstitial blood vessels, which increased in number and size at 20 cm CVRL and decreased afterwards. By TEM, three types of Leydig cells were observed at 14.5 cm CVRL as stem Leydig cells (Leydig cell A), progenitor Leydig cells (Leydig cell B) and immature Leydig cells (Leydig cell C). Leydig cell A had elongated nucleus with a very thin rim of cytoplasm. Leydig cell B had oval nucleus with comparatively larger amount of cytoplasm. Leydig cell C had adult Leydig cell like features and had round to oval nucleus with lager amount of cytoplasm. The cytoplasm of these cells contained mitochondria with elongated cristae, Golgi apparatus and very few lipid inclusions. The number and size of Leydig cells increased during early prenatal life.

A Review of Toxoplasmosis and Neosporosis in Water Buffalo (Bubalus bubalis).

Toxoplasmosis and neosporosis are diseases with worldwide distribution that are associated with reproductive problems in livestock and responsible for economic losses. This review presents an overview of the current knowledge relative to these diseases in water buffalo (Bubalus bubalis). In general, buffalo are considered resistant to clinical toxoplasmosis because there are studies only reporting serological evidence of natural infection in these animals. Studies have described age, poor hygienic status of the farm, and presence of cats as risk factors for the development of Toxoplasma gondii infection in buffalo. It must be highlighted that buffalo meat, which does not receive adequate freezing treatment, could be a potential source for toxoplasmic human infection as well as the importance of raw buffalo milk in the transmission of toxoplasmosis to human beings. Neospora caninum is considered one of the major causes of abortion and responsible for huge economic losses in cattle. Vertical transmission is the main route to infect calves, and is responsible for maintaining the parasite within a herd. In buffalo, vertical transmission is also described; moreover, although there are indications that N. caninum may be associated with abortion in dairy buffalo, the reproductive importance of neosporosis is apparently lower in buffalo relative to cattle. Most studies have identified a higher time of exposition to N. caninum oocysts relative to age. The household system was also described as a risk factor for infection, possibly due to persistent contact between the home-raised buffalo and canids. The fetal immune competence of buffalo is similar to bovine, and buffalo fetus are highly susceptible to infection during the first trimester of pregnancy, indicating that N. caninum may be an abortigenic agent in buffaloes. Alternatively, it is interesting to note there is evidence that the inflammatory response in pregnant buffalo infected with N. caninum is mild enough to avoid abortion in most cases. It is proposed that the possible transmission of toxoplasmosis through unprocessed milk and buffalo meat may occur, which is important in terms of public health. Additionally, there is strong evidence to suggest that N. caninum may be associated with abortion in buffalo.

Transposon mediated reprogramming of buffalo fetal fibroblasts to induced pluripotent stem cells in feeder free culture conditions

Commonly, induced pluripotent stem (iPS) cells are generated by viral transduction of four core reprogramming genes, but recent evidences suggest that slightly different combination of transcription factors improve the efficiency and quality of generated iPS cells. However, vectors like retro- and lentiviral may cause insertional mutagenesis due to its integrating ability. Hence, alternate methods with safety concerns are needed to be investigated. Therefore, the present study was undertaken to reprogram buffalo fibroblasts using non-viral piggyBac (PB) transposon mediated transfer of six transcription factors. To generate buffalo iPS cells, fibroblasts were isolated from buffalo fetus at passage 2. The cells were co-electroporated with a PB transposon having CAGGS promoter driven cassette of Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 transcription factors separated by self-cleaving 2A peptide and a helper plasmid pCMV-PB transposase. After 12–14 days post electroporation, fibroblast cells morphology was observed to change to round structures which formed loose aggregates of cells on day 18. Putative iPS cell colonies were propagated in feeder free system and characterized through expression of pluripotency markers such as alkaline phosphatase, SSEA-1, SSEA-4, SSEA-5, TRA-1-81, Oct4, Nanog and Sox2 and endogenous genes supported the stemness property of the generated cells. These cells differentiated in vitro to form embryoid bodies and were found to express three germ layers markers. In conclusion, generation of buffalo iPS cells using transposon system provides insights into viral-free iPS technology which will facilitate genetic modification of the buffalo genome and help in the production of transgenic animals using genetically modified iPS cells.

Prenatal development of retina in buffalo (Bubalus bubalis).

The development of retina in Indian buffalo (Bubalus bubalis) has not been reported previously. The aim of the present study was therefore to report the major landmarks and the time course in the development of retina. Serial histological sections of Indian buffalo embryos and foetuses were used as group1 (<20.0cm CVRL), group2 (>20.0 but <40.0cm CVRL) and group3 (>40.0cm CVRL). Age estimation was made on the basis of crown vertebral-rump length (CVRL), which ranged between 36 and 286days (1.6-94.0cm). The retina in Indian buffalo was developed in a similar manner to that of the other mammals with the principal differences in the time of occurrence of various layers of this nervous tunic. In 36days (1.6cm stage), the foetal retina was composed of pigmented layer and the layer of neuroblasts. Differentiation of layers was first observed in 47days (4.0cm CVRL) which became prominent in 52days (5.1cm stage). At 120days (20.5cm stage), the differentiation of inner plexiform layer and inner nuclear layer was evident. At 143days (31.0cm) foetal age, the faint line in neuroblastic layer was the first evidence of the future outer plexiform layer. In foetuses of group III, the retina was comprised of all 10 layers (eight cell layers and two membranes) viz. pigmented epithelium, layer of rods and cones, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, layer of nerve fibres and the inner limiting membrane.

Efficient Generation of Transgenic Buffalos (Bubalus bubalis) by Nuclear Transfer of Fetal Fibroblasts Expressing Enhanced Green Fluorescent Protein

The possibility of producing transgenic cloned buffalos by nuclear transfer of fetal fibroblasts expressing enhanced green fluorescent protein (EGFP) was explored in this study. When buffalo fetal fibroblasts (BFFs) isolated from a male buffalo fetus were transfected with pEGFP-N1 (EGFP is driven by CMV and Neo is driven by SV-40) by means of electroporation, Lipofectamine-LTX and X-tremeGENE, the transfection efficiency of electroporation (35.5%) was higher than Lipofectamine-LTX (11.7%) and X-tremeGENE (25.4%, P < 0.05). When BFFs were transfected by means of electroporation, more embryos from BFFs transfected with pEGFP-IRES-Neo (EGFP and Neo are driven by promoter of human elongation factor) cleaved and developed to blastocysts (21.6%) compared to BFFs transfected with pEGFP-N1 (16.4%, P < 0.05). A total of 72 blastocysts were transferred into 36 recipients and six recipients became pregnant. In the end of gestation, the pregnant recipients delivered six healthy calves and one stillborn calf. These calves were confirmed to be derived from the transgenic cells by Southern blot and microsatellite analysis. These results indicate that electroporation is more efficient than lipofection in transfecting exogenous DNA into BFFs and transgenic buffalos can be produced effectively by nuclear transfer of BFFs transfected with pEGFP-IRES-Neo.

Identification of Primary Ossification Centers in the Skull of Buffalo Fetus by Modified Alizarin Red-S Method

Identification of Primary Ossification Centers in the Skull of Buffalo Fetus by Modified Alizarin Red-S Method

Enzyme histochemistry of cecal lymphoid tissue during prenatal period of buffalo.

Aim:This study was designed to elucidate the histoenzymic distribution of enzymes, i.e., phosphatases, oxidoreductases, dehydrogenases, and diaphorases in cecal lymphoid tissue during its development in the prenatal period.Materials and Methods:The study was conducted on cecum of 15 buffalo fetuses ranging from 16 cm curved crown-rump length (CVRL) (100 days) to 100 cm CVRL (full term). The fetuses were categorized into three groups based on their CVRL.Results:In Group I, the distribution of enzymes was uniformly weak in developing villi-like projections in cecum and completely absent from submucosa. In Group II, the enzymes showed a moderate to strong activity in epithelium lining tunica mucosa which progressively decreased as the fetus progresses toward late gestational age. However, the intense activity of enzymes was observed in developing lymphoid tissue in this group. In Group III, distribution of enzymes reduced in tunica mucosa of cecum with advancing age, whereas the intense activity was noticed in the developed lymphoid tissue complex.Conclusion:The distribution of enzymes was completely absent from submucosal region in cecum of Group I as there was no lymphoid tissue development at this age. In Group II, the enzymes showed a moderate to strong activity in epithelium lining tunica mucosa which progressively decreased toward late gestational age but an intense activity was observed in developing lymphoid tissue. In Group III, distribution of enzymes reduced in tunica mucosa with advancing age with intense activity noticed in the developed lymphoid tissue complex.

Histoenzymic studies on sublingual salivary gland of buffalo during prenatal development

The histoenzymic studies on sublingual salivary gland of 15 buffalo foetuses ranging from 11 cm CVRL (78 days) to 100 cm CVRL (full term) were conducted. The foetuses were categorized into three groups based on their curved crown rump length (CVRL). At 11-19 cm CVRL (78-114 days), a weak activity of phosphatases and oxidoreductases was observed in the acinar cells and ductular epithelium. Between 28-37 cm CVRL (136-157 days), a weak to moderate diffused granular alkaline phosphatase (AKPase) activity was observed in the mucous acini whereas oxidoreductases showed moderate activity. The enzyme activity was progressively increased with advancement of the gestation period. AKPase activity was more in the lumen of acini and along the intercellular canaliculi at 42-100 cm of CVRL (168 days- full term). Large ducts exhibited strong activity for oxidoreductases indicating increased metabolic activity of cells.

Ultrastructural changes in the sublingual salivary gland of prenatal buffalo (Bubalus bubalis)

Aim:The present study was aimed to elucidate ultrastructural changes in the development of sublingual salivary gland of buffalo during prenatal life.Materials and Methods:The study was carried out on sublingual salivary gland of 36 buffalo fetuses ranging from 13.2 cm curved crown-rump length (CVRL) (88th day) to full term. The fetuses were categorized into three groups based on their CVRL.Results:The cells lining the terminal tubules were undifferentiated with poorly developed cytoplasmic organelles but lacked secretory granules (SGs) at 13.2 cm CVRL (88th day). The SGs appeared first in the form of membrane-bound secretory vesicles with homogeneous electron-dense as well as electron-lucent contents at 21.2 cm CVRL (122nd day); however, mucous acinar cells contained electron-lucent granules, while serous secretory cells as well as serous demilunes showed electron-dense granules at 34 cm CVRL (150th day) of prenatal life. At 53.5 cm CVRL (194th day), both mucous and serous acini were differentiated by the density of SGs.Conclusion:The cytoplasm of acinar cells was filled with mitochondria, rough endoplasmic reticulum, and Golgi profiles in mid and late fetal age groups. The SGs were increased in number during the late fetal age group. The myoepithelial cells (MECs) were located at the base of the acinar cells as well as intercalated and striated ducts and were stellate in shape. The ultrastructure of MEC revealed a parallel stream of myofilaments in the cytoplasm and its processes. The mucous cells were predominantly present in the sublingual salivary gland and were pyramidal in shape.

Light and Electron Microscopic Studies on Prenatal Differentiation of Exocrine Pancreas in Buffalo.

The study was conducted on pancreas of 24 buffalo fetuses collected from abattoir and Veterinary clinics, GADVASU, Ludhiana. The buffalo fetuses were divided into three groups after measuring their CVRL, namely, group I (CVRL between 0 and 20 cm), group II (CVRL above 20 cm and up to 40 cm), and group III (CVRL above 40 cm) and their approximate age was calculated. The tissues were processed for light and ultrastructural studies. In group I, at 1.2 cm CVRL (34 days), the pancreas comprised tubules and solid nest of undifferentiated epithelial cells. At 7.5 cm CVRL (63 days) acinar cells with zymogen granules were observed. These acinar cells varied in shape from columnar to pyramidal. At 12.8 cm CVRL (86 days), parenchyma began to organize into lobes and lobules. The centroacinar cells were observed at 12.8 cm CVRL (86 days). In group II, at 28.3 cm CVRL (137 days), there was extensive branching of tubules that resulted in highly branched ductal tree connecting exocrine secretary units to the duct system. The interlobular and intralobular ducts were well observed at this age yet the intercalated ducts were not completely developed. In group III, exocrine pancreas showed a massive growth at 48 cm CVRL (182 days) with distinct pancreatic lobes and lobules. At 54 cm CVRL (195 days), well developed pancreatic architecture was seen with the presence of extensive development of exocrine part organized in lobes and lobules with interlobular and intralobular ducts whereas the intercalated ducts were observed in 80 cm CVRL (254 days).