Successful artificial insemination in the Asian elephant (Elephas maximus) using chilled and frozen-thawed semen

<p>Abstract</p> <p>Background</p> <p>Artificial insemination (AI) using frozen-thawed semen is well established and routinely used for breeding in various mammalian species. However, there is no report of the birth of elephant calves following AI with frozen-thawed semen. The objective of the present study was to investigate the fertilizing ability of chilled and frozen-thawed semen in the Asian elephant following artificial insemination (AI).</p> <p>Methods</p> <p>Semen samples were collected by from 8 bulls (age range, 12-to 42-years) by manual stimulation. Semen with high quality were either cooled to 4°C or frozen in liquid nitrogen (-196°C) before being used for AI. Blood samples collected from ten elephant females (age range, 12-to 52-years) were assessed for estrus cycle and elephants with normal cycling were used for AI. Artificial insemination series were conducted during 2003 to 2008; 55 and 2 AI trials were conducted using frozen-thawed and chilled semen, respectively. Pregnancy was detected using transrectal ultrasonography and serum progestagen measurement.</p> <p>Results</p> <p>One female (Khod) inseminated with chilled semen became pregnant and gave birth in 2007. The gestation length was 663 days and the sex of the elephant calf was male. One female (Sao) inseminated with frozen-thawed semen showed signs of pregnancy by increasing progestagen levels and a fetus was observed for 5 months by transrectal ultrasonography.</p> <p>Conclusion</p> <p>This is the first report showing pregnancy following AI with frozen-thawed semen in the Asian elephant. Successful AI in the Asian elephant using either chilled or frozen-thawed semen is a stepping stone towards applying this technology for genetic improvement of the elephant population.</p

Analisis Perubahan Garis Pantai Di Pantai Cacalan, Banyuwangi, Jawa Timur Dengan Metode Numerik

Pantai Cacalan merupakan salah satu destinasi wisata yang ada di Kabupatan Banyuwangi. Pantai Cacalan memang tidak sepopuler pantai lain yang ada di Banyuwangi. Pantai Cacalan memiliki garis pantai memanjang sekitar 1,2 km. Pantai Cacalan memiliki hamparan pasir hitam ke abu-abuan dan memiliki ombak yang relatif kecil. Garis pantai adalah daerah perbatasan antara daratan dan lautan. Garis pantai merupakan daerah yang masih dipengaruhi oleh pasang surut, baik pasang tertinggi dan surut terrendah Ada dua macam perubahan garis pantai yang pertama garis pantai mengalami kemunduran hal ini biasanya disebut dengan abrasi. Sedangkan apabila garis pantai semakin menjorok kelautan disebut dengan sedimentasi. Penelitian ini bertujuan untuk mengetahui perubahan garis pantai dan karakteristik sedimen yang ada di Pantai Cacalan, Banyuwangi. Penelitian Analisis Perubahan Pantai di Pantai Cacalan, Banyuwangi akan mengunakan metode numerik dengan rumus CERC (Coastal Engineering Research Center). Ada dua data yang akan digunakan dalam penelitian ini yang pertama ialah data primer berupa tracking garis pantai, mengukur kemiringan pantai, pengambilan sedimen dan pengambilan data gelombang. Sedangkan data sekunder didapatkan dari pengambilan citra garis pantai yang terdahulu oleh Google Earth dan data pasang surut dari PT. Pelabuhan Indonesia III Tanjung Wangi. Perubahan garis pantai di Pantai Cacalan, Banyuwangi yang telah dilakukan dari dua metode yang berbeda yaitu melalui citra satelit dan perhitungan numerik dengan rumus CERC. Perubahan garis pantai Cacalan pada tahun 2011 sampai 2014 terjadi abrasi dengan nilai perubahan sebesar - 5,58 meter atau -1,86 meter per tahun, begitu juga pada tahun 2014 sampai 2018 Pantai Cacalan mengalami abrasi dengan nilai perubahan sebesar -12,22 meter atau -3,05 meter per tahun. Sedangkan selama tahun 2011 sampai 2018 terjadi abrasi dengan nilai perubahan sebesar -18,11 atau -2,59 meter per tahun. Hasil perhitungan numerik dengan rumus CERC garis pantai Cacalan mengalami abrasi pada 1 tahun mendatang dengan nilai -2.25 meter pertahun

Effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes

<p>Abstract</p> <p>Background</p> <p>Cryopreservation of oocytes, which is an interesting procedure to conserve female gametes, is an essential part of reproductive biotechnology. The objective of the present study was to investigate the effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes.</p> <p>Methods</p> <p>Immature oocytes (germinal vesicles) isolated from ovaries of normal bitches (> 6 months of age) were either vitrified in open pulled straw (OPS) using 20% ethylene glycol (EG) and 20% dimethyl sulfoxide (DMSO) as vitrification solution or exposed to vitrification solution without subjected to liquid nitrogen. After warming, oocytes were investigated for nuclear maturation following in vitro maturation (IVM), ultrastructural changes using transmission electron microscopy (TEM) and gene expression using RT-PCR. Fresh immature oocytes were used as the control group.</p> <p>Results</p> <p>The rate of resumption of meiosis in vitrified-warmed oocytes (53.4%) was significantly (P < 0.05) lower than those of control (93.8%) and exposure (91.4%) groups. However, there were no statistically significant differences among groups in the rates of GV oocytes reaching the maturation stage (metaphase II, MII). The ultrastructural alterations revealed by TEM showed that cortical granules, mitochondria, lipid droplets and smooth endoplasmic reticulum (SER) were affected by vitrification procedures. RT-PCR analysis for gene expression revealed no differences in HSP70, Dnmt1, SOD1 and BAX genes among groups, whereas Bcl2 was strongly expressed in vitrified-warmed group when compared to the control.</p> <p>Conclusion</p> <p>Immature canine oocytes were successfully cryopreserved, resumed meiosis and developed to the MII stage. The information obtained in this study is crucial for the development of an effective method to cryopreserve canine oocytes for establishment of genetic banks of endangered canid species.</p

Development of swamp buffalo (Bubalus bubalis) embryos after parthenogenetic activation and nuclear transfer using serum fed or starved fetal fibroblasts

The knowledge of oocyte activation and somatic cell nuclear transfer in the swamp buffalo (Bubalus bubalis) is extremely rare. The objectives of this study were the following: (1) to investigate the various activation treatments on the parthenogenetic development of buffalo oocytes, (2) to examine the events of nuclear remodeling and in the in vitro development of cloned buffalo embryos reconstructed with serum fed or starved fetal fibroblats, and (3) to investigate the in vivo development of cloned embryos derived from serum fed or starved cells after transfer into the recipients. The rates of cleavage and blastocyst development were found to be significantly higher (P < 0.05) when the oocytes were activated by the combination treatment of calcium ionophore (A23187) or ethanol followed by 6-DMAP than those activated by electrical pulses and 6-DMAP or other single treatments. Flow cytometric analysis revealed that the percentage in the G0/G1 phase in serum starved cells was significantly (P < 0.05) higher than that in serum fed cells (88.8 ± 6.2 vs. 68.2 ± 2.6). At 1 h post fusion (hpf), most of the transferred nuclei (71%) from serum fed cells did not change in size, and the nuclear envelope remained intact, whereas 29% underwent NEBD and PCC. When serum starved cells were used, 83% of the transferred nuclei underwent NEBD and PCC whereas 17% remained intact. The nuclear swelling and pronucleus (PN) formation were observed at 2–4 and 12 h post activation (hpa), respectively. The remodeled nuclei underwent mitotic division and developed to the 2-cell stage within 18–24 hpa. Fifty-five percent of oocytes reconstructed with serum fed cells were 2PN and 45% were 1PN, whereas 79% of the embryos reconstructed from starved cells were 1PN and 21% were 2PN. The percentage of blastocyst development of the embryos derived from starved cells was higher than that from the serum fed cells (35% vs. 21%, P < 0.05). Pregnancy was detected after the transfer of cloned blastocysts into the recipients but no recipients supported the development to term. The results of this work can be used to establish effective activation protocols for buffalo oocytes which can be used during nuclear transfer experiments

Seminal Plasma MDA Concentrations Correlating Negatively with Semen Quality in Asian elephants บทคั ดย่ อ ความสั มพั นธ์ แบบแปรผกผั นระหว่ างระดั บของ MDA ในน้ ํ าเลี ้ ยงเซลล์ อสุ จิ กั บคุ ณภาพน้ ํ าเชื ้ อช้ าง เอเชี ย สิ ทธวี ร์ ทองทิ พย์ ศิ ริ เดช 1,

Abstract The aim of this study was to determine whether lipid peroxidation is correlated with semen quality in Asian elephant bulls. Malondialdehyde (MDA) in seminal plasma from ejaculates with varying percentages of progressive motility was measured using Thiobarbituric Acid method. Correlation between the MDA levels and percentages of progressive motility and normal morphology were performed. Results revealed that the MDA levels were significantly negative, which correlated (p&lt;0.05) with the percentages of progressive motility and normal morphology (R=0.2131 and 0.1685, respectively). The results also showed that the MDA levels were significantly difference between each bull (p&lt;0.01). Furthermore, when the ejaculates were grouped according to motility scores into two groups; low-(&lt;40%) and high-percentage of progressive motility (&gt;40%), a significant difference was detected between the MDA means (±SD) of the low-(20.7±11.4 nmol/ml) and high-percentages of progressive motility (14.4±7.8 nmol/ml) groups. The results obtained from this study suggested that MDA could be a potential parameter applicable for the assessment of elephant semen quality. It could as well be deduced from this study that oxidative stress might play a key role in low fertility due to poor semen quality in captive male elephants. This data provides beneficial information to better understanding of elephant reproduction in captivity

Potential factors affecting semen quality in the Asian elephant (Elephas maximus)

Abstract Background One of the major obstacles in using artificial insemination to manage genetics of elephant population in captivity is the large variations in semen quality among ejaculates within the same and among individuals. The objectives of this study were to determine the influences of (1) age (2) seasonality (3) and circulating testosterone (SrTest), triiodothyronine (SrT3) and tetraiodothyronine (SrT4), as well as seminal (4) testosterone (SpTest), zinc (SpZn) and protein (SpTP) on semen quality in the Asian elephant Methods Analyses, including motility, viability and morphology were performed in semen samples collected twice monthly from 13 elephant bulls (age range, 10-to 72-years) by manual stimulation between July 2004 and June 2005. Serum samples obtained monthly were assessed for SrTest, SrT3, SrT4, and seminal plasma samples were evaluated for, SpTest, SpZn and SpTP. Results The highest semen quality was observed at age 23 to 43 years. Percentages of progressive motility and viable sperm were lowest at age 51 to 70 years (P Conclusion This study indicates that age and seasonality had influence on semen characteristics in the Asian elephant. The knowledge obtained in this study will improve our understanding of the reproductive biology of this species.</p

Missing and overexpressing proteins in domestic cat oocytes following vitrification and in vitro maturation as revealed by proteomic analysis

Abstract Background The domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family. Methods Immature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC–MS/MS. Results A total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented. Conclusions The identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species

Assessment of viability and acrosomal status of Asian elephant (Elephas maximus) sperm after treatment with calcium ionophore and heparin

Knowledge about the acrosomal status of Asian elephant (Elephas maximus) sperm is extremely limited. The objective of this study was to evaluate the viability and acrosomal status of Asian elephant sperm following induction by calcium ionophore and heparin using propidium iodide (PI) and fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA). Semen samples were collected from elephant bulls by manual stimulation. Semen was diluted with extender, cooled to 4°C and transported to a laboratory for the experiment. Sperm cells were incubated in modified Tyrode's medium containing either 1mM calcium ionophore or 10 mg/mℓ heparin for 5 h at 39°C. Sperm recovered at the onset (0 h), 1, 2, 3, 4 and 5 h of incubation were simultaneously assessed for the viability and acrosomal status using dual staining of FITC-PNA and PI. Results were confirmed by transmission electron microscopy. A progressive increase in the proportion of live-acrosome reacted sperm was observed within 3 h of incubation in both treatment groups which slightly decreased at 4 to5 h of incubation. At 1 to 3 h of incubation, the percentage of live-acrosome reacted sperm induced by calcium ionophore was higher (P < 0.05) than those induced by heparin and the control. However, there were no statistical differences at 4 to 5 h of incubation. A progressive reduction of the percentage of motile sperm was observed in the control as well as both treatment groups. Sperm motility decreased sharply when they were incubated in calcium ionophore compared with incubation in heparin and control groups. These results indicate that the occurrence of live-acrosome reacted sperm in the Asian elephant was induced by calcium ionophore at a rate higher than that induced by heparin