Camel Oocytes Research Articles

The Impact of Follicular Fluid on the in vitro Maturation of Camel Oocytes

Background: The objective of this research is to find out how much of an impact adding camel follicular fluid (CFF) to camel oocytes in vitro maturation (IVM) medium has on getting meiosis back on track. Methods: Ovaries from camels were obtained at the central slaughterhouse in Riyadh, Kingdom of Saudi Arabia (KSA). Oocytes were extracted from follicles ranging in size from 2 to 8 mm. Camel oocytes were cultivated in a maturation medium containing varying amounts of camel follicular fluid: 0% (control), 2.5% and 5% for a duration of 32-36 hours. Result: According to the result, the maturation rate of oocytes was considerably (p.05) lower in the 2.5% CFF and 5% CFF groups compared to the control group. It is advised that greater CFF concentrations of camel oocytes be added to the IVM culture in order to improve nuclear and cytoplasmic maturation and boost oocyte competency.

Antibiotic Resistance Microbial Contamination During In-vitro Maturation of Bovine and Camel Oocytes: Causes and Management

Antibiotic Resistance Microbial Contamination During In-vitro Maturation of Bovine and Camel Oocytes: Causes and Management

In vitro maturation (IVM) of camel oocytes using different maturation media

In vitro maturation (IVM) of camel oocytes using different maturation media

Influences of glycine supplementation during vitrification on the developmental potential of vitrified/warmed immature dromedary camel oocytes.

Oocytes experience detrimental osmotic stress during vitrification and warming procedures because of the osmolality imbalance between the vitrification-warming fluids and the intracellular environment. Cellular osmotic homeostasis can be preserved by glycine, a powerful osmolyte with antioxidant properties. We aimed to examine the influences of supplementing glycine to the vitrification solutions (VS) on the developmental potential of vitrified/warmed immature dromedary camel oocytes following IVM/IVF and in vitro embryo culture (IVC). Cumulus oocyte complexes (COCs) were collected from dromedary camel ovaries and randomly allocated into two groups namely control (oocytes subjected directly to IVM) and vitrified (COCs were vitrified into VS supplemented with 0.0, 0.5, 1.0 or 2.0 mM glycine). For vitrification, COCs were equilibrated for 3 min in 12.5% ethylene glycol; EG plus 12.5% dimethyl sulfoxide; DMS and then they were vitrified for 60 s in VS composed of 25% EG + 25% DMSO using solid surface vitrification (SSV). Warming of vitrified oocytes was conducted in decreasing concentrations of trehalose solution. Following vitrification and warming, the morphologically viable oocytes were subjected to IVM for 36 h. Matured oocytes were then fertilized in vitro by epididymal spermatozoa and cultured for seven days. The results showed that the percentage of viable oocytes assessed by trypan blue stain was significantly higher (p ≤ .05) in the 1.0 mM glycine-supplemented group than 0.0- and 2.0-mM glycine-supplemented ones (90.0 % vs. 80.0% and 76.6%, respectively). However, no significant difference was observed between 0.5 mM glycine and other vitrified groups. Nuclear maturation rates, cleavage (48-h post-insemination; pi) and blastocyst rate (7-days pi) were significantly lower in vitrified groups than control ones (p ≤ .05). Among vitrified groups, these parameters were the highest in the 1.0 mM glycine-supplemented group. Taken together, supplementation of vitrification solutions with 1.0 mM glycine could enhance the developmental potential of vitrified/warmed immature dromedary camel oocytes.

Insulin-like growth factor-1 improves in vitro meiotic resumption of dromedary camel (Camelus Dromedarius) oocytes.

Despite relatively high maturation rate of in vitro matured oocytes in the dromedary camel, however, blastocyst production is very low after in vitro fertilization (IVF). Herein, the influences of oocyte collection method (follicular aspiration vs slicing; Experiment I), the addition of Insulin-like growth factor I (IGF-I) to the maturation medium (Experiment II) on in vitro maturation (IVM) of oocyte were investigated. Although the nuclear maturation did not differ regardless of collecting method, follicular aspiration led to lower degeneration rates than those in controls (P < 0.05). The percentages of oocytes at MII were greater in the presence of IGF-1 than in its absence (71.9% vs 48.4%, respectively, P<0.05). Additionally, the percentages of degenerated oocytes were higher in the control group compared to oocytes cultured in the presence of IGF-I (23.6% vs 10.4%, respectively, P<0.05). IGF-I treatment improved the quality of MII matured oocytes as evidenced by the decrease of cathepsin B (CTSB) activity, a marker of poor quality oocytes, when compared to control ones (P < 0.05). In conclusion, follicular aspiration decreased the degeneration rate; however, it had no effect on completion of maturation. IGF-I enhanced the IVM of oocyte and decreased degeneration rate.

Effects of Ascorbic Acid on Maturation Rate, Morphology, and Gene Expression of Vitrified In Vitro Matured Dromedary Camel Oocytes

In vitro embryo generation, cryopreservation, and embryo transfer are examples of assisted reproductive technologies that can be used to improve camel genetic performance and fertility. The aim of this study was to investigate the impact of ascorbic acid supplementation to in vitro maturation media on the maturation rate, morphology, and gene expression of fresh and vitrified in vitro matured dromedary camel oocytes. In the current study, 810 oocytes of excellent and good quality were in vitro matured in maturation medium (TCM-199 + 10 ug/ml follicle stimulated hormone + 10% fetal calf serum + 100 IU/ml Pregnant mare serum + 50 μg/ml gentamycin) without any additives to act as a control group (C) and with 50 μg/ml ascorbic acid group (AA) and incubation in a CO2 incubator (38.5 ̊C, 5% CO2, 20% O2 and 95% humidity) for 40 hours. In vitro matured dromedary camel oocytes with the first polar body (n = 210) in C group and AA group (n = 250) were placed in basic medium (BM) and then placed in vitrification solution1 (VS1) for one minute, followed by the transfer of oocytes to VS2 (double concentration of VS1, containing 20% Ethyl Glycol (EG) and +20% Dimethyl sulfoxide) for 30 seconds. Oocytes were then loaded into sterile 0.25 ml straws and stored in liquid nitrogen for 7-10 days. The normal fresh and vitrified /thawed in vitro matured dromedary camel oocytes were kept in RNA later at a -80°C freezer for gene expression analysis. The maturation rate of dromedary camel oocytes in the in vitro matured AA group was significantly higher than that of the C group. The percentage of normally recovered vitrified/thawed oocytes was higher in the in vitro matured with ascorbic acid (VAA) than in the control (VC) group. The expression pattern of the SOD1 gene and GDF9 gene was upregulated in fresh AA and VAA groups than in the fresh C and VC groups. The profile of the SOD1 gene was more abundant in the vitrified/thawed oocytes VAA group than in the VC group. All vitrified/thawed groups, whether control or ascorbic acid supplemented, had lower levels of SOD1, GDF9, and BMP15 expression, compared to the fresh groups. In conclusion, the supplementation of the maturation medium with ascorbic acid has an increased maturation rate, and normal morphology of vitrified/ thawed oocytes which was linked with upregulation of SOD1, GDF9 genes expression.

Impact of Presence or Absence of Trehalose during Vitrification on Viability and Development of Vitrified/Warmed Immature Dromedary Camel Oocytes

Vitrification of immature oocytes at the germinal vesicle (GV) stage is important to preserve female gametes. The standard formula for vitrification solutions has long been a debate. Herein, we investigated the effect of the presence or absence of trehalose in vitrification solution on viability, in vitro maturation (IVM) rates, and development of vitrified/warmed immature dromedary camel oocytes. Cumulus oocyte complexes (COCs) obtained at slaughter from the ovaries of mature she-camels were randomly allocated into three groups; namely, control group, oocytes were directly subjected to IVM without vitrification, vitrification solution 1 (VS1) group, oocytes were vitrified in a solution composed of 25% ethylene glycol (EG) plus 25% dimethyl sulfoxide (DMSO) + 0.5 M trehalose; and vitrification solution 2 (VS2) group, oocytes were vitrified in a solution composed of 25% EG plus 25% DMSO. Vitrification of COCs was conducted by open pulled straws (OPS) method. Following vitrification and warming, morphologically viable oocytes were matured in vitro for 36 h. COCs were then fertilized and cultured in vitro for 7 days. The percentage of viable oocytes was significantly higher (P 0.05) in VS2 than VS1 group (80.0% vs. 63.3%, respectively). Nuclear maturation, cleavage (48 h post-insemination; pi), and blastocyst rates (7 days pi) were significantly higher (P camel oocytes in trehalose-free solution (VS2) was more advantageous than that in trehalose supplemented media since it did not reduce viability and development.

Effects of Antioxidants on Developmental Competence of Matured Dromedary Camel Oocytes

The purpose of this work was to look into the impact of antioxidants on the viability and mitochondrial distribution of dromedary camel oocytes, through 1) Study the effect of vitamin C (ascorbic acid) and melatonin on the maturation rate and fertilization rate of dromedary camel oocytes, 2) Study the impact of vitamin C and melatonin on the viability, mitochondrial distribution, and intensity of in-vitro matured dromedary camel oocytes. Camel oocytes were aspirated from ovaries. Oocytes of excellent and good quality were in-vitro matured using in vitro maturation media (TCM-199 + 10 ug/ml FSH + 10%fetal calf serum + 100 IU/ml Pregnant mare serum + 50 mg/ml gentamycin) without (control group) or with antioxidant groups (50 µg/ml vitamin C or 100 µM/ml melatonin) and incubated in CO2 incubator (38.5 C, 5% CO2, 20% O2 and 95% humidity) for 40 hours. In vitro matured oocytes with 1st polar body were either in vitro fertilized using epididymal spermatozoa for detection the fertilization rate or stained with Hoechst 33342 (bis-Benzimide H33258) dye and MitoTracker red dye for detection the viability and mitochondrial distribution and intensity using confocal microscope. Results showed that, the nuclear maturation rate of dromedary camel oocytes in vitro matured with antioxidants were significantly (P˂0.001) higher than the control group. The fertilization rate of in vitro matured oocytes in the control group (56.26 ± 0.63) was lower than vitamin C and melatonin group (70.88 ± 0.87 and 76.63 ± 0.82, respectively). The mitochondrial intensity was significantly increased in the vitamin C (118.3 ± 1.13) and melatonin (231.1 ± 4.06) groups when compared with control group (102.5 ± 2.16). The melatonin group was significantly higher in peripheral mitochondrial distribution when compared with control and vitamin C groups, while the diffused distribution of mitochondria was markedly greater in the vitamin C and control group when compared with melatonin group. In-conclusion, Supplementation of vitamin C or melatonin on in vitro maturation media were significantly improved the maturation rate and fertilization rate through enhancing the mitochondrial intensity and distribution in Dromedary camel oocytes.

Development and pregnancy rates of Camelus dromedarius-cloned embryos derived from in vivo- and in vitro-matured oocytes.

ObjectiveThe present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius.MethodsCamelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method.ResultsThe collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitro-matured oocytes.ConclusionIn this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.

Effect of melatonin supplementation during IVM of dromedary camel oocytes (Camelus dromedarius) on their maturation, fertilization, and developmental rates in vitro

The positive impact of melatonin on in vitro embryo production (IVEP) has been reported in many domestic species; however, no studies have been carried out in camelids. We aimed to evaluate the effects of melatonin supplementation in maturation media on in vitro maturation, fertilization, and preimplantation embryo development of dromedary camel oocytes (experiment 1). We also evaluated the concentrations of total antioxidant capacity (TAC), and malondialdehyde (MDA) in the IVM spent medium in relation to melatonin supplementation. Cumulus oocyte complexes (COCs) were cultured in in vitro maturation media (IVM) supplemented with either 0.0, 25.0, 50.0 or 75.0 μM of melatonin for 30 h. Matured oocytes were then fertilized in vitro with epididymal camel spermatozoa. Following IVF, the resulting embryos were cultured in vitro for seven days. The percentage of maturation, fertilization, cleavage, and embryo developmental rates (morula and blastocyst) was recorded (experiment 1). TAC and MDA levels in the IVM spent maturation media were also evaluated at 30 h post-IVM (experiment 2). The results showed that supplementation of IVM media with 25 μM melatonin significantly improved oocyte nuclear maturation, fertilization (18 h post-insemination; pi), cleavage (day 3 pi), morula (day 5 pi) and blastocyst (day 7 pi) rates as compared with the controls and other melatonin-supplemented groups. Furthermore, the TAC in the IVM spent media was significantly increased (P < 0.05) in 25 μM melatonin supplemented groups than those supplemented with 0.0, 50.0, 75.0 μM melatonin. However, the concentration of MDA was significantly lower (P < 0.05) in IVM media supplemented with 25.0 μM of melatonin when compared with the control and other treatment groups. In conclusion, supplementation of IVM medium with 25 μM of melatonin could enhance the in vitro developmental capacity of dromedary camel oocytes.

Effect of Heat Stress on Developmental Competence of In Vitro Matured Oocytes of Camelus Dromedaries with Different Qualities

The deleterious effect of heat stress on cumulus-oocytes complexes (COCs) competence is well recognized in different livestock species. Therefore, the present study aimed to investigate the effect of physiologically relevant heat stress on the developmental competence of camel COCs during in vitro maturation (IVM). A total of 1548 COCs were divided into six groups in this study. The groups were named K1 and K2 representing good and low-quality COCs incubated at 38.5oC for 30 hours. While K3 and k4 represent good and low-quality COCs exposed to 41oC for the first 6 hours of IVM. Finally, K5 and k6 represent the groups of good and low-quality COCs exposed to 42oC for the first 6 hours of IVM. After exposure of COCs to heat stress at 41°C and 42°C during the first 6 hours of in vitro maturation, the COCs were incubated at 38.5°C for 24 hours of IVM. The in vitro matured COCs were activated to cleave using ethanol followed by 4 mM 6-DMAP and developed embryos were cultured in vitro for 7 days post parthenogenetic activation. The results of this study indicated that heat stress at 42oC significantly decreased the Pb (polar body) extrusion rate in K4 and K6, compared to other groups. Additionally, the embryo cleavage rate was significantly lower for good and low-quality oocytes exposed to heat stress (K2, K3, K4, K5, and K6), compared to good quality COCs of the control group (K1). The cleavage rate was lower for low quality (K2; 63 ± 1.28) than good quality COCs (K1; 53 ± 1.85). The percentages of oocytes that developed to the blastocyst stage were lower for K2, K3, K4, K5, and K6 than K1. Moreover, the blastocyst rate was lower for K2 (9 ± 0.22) than K1 (15 ± 0.22). The results of this study indicated that exposure of camel oocytes to heat stress for 6 hours during in vitro maturation severely reduced extrusion of polar body, cleavage, and blastocyst rates. The low-quality camel COCs were reduced developmental capacity than good quality oocytes.

Vitrification of camel oocytes transiently impacts mitochondrial functions without affecting the developmental potential after intracytoplasmic sperm injection and parthenogenetic activation.

Oocyte vitrification preserves the female genetic resources of elite dromedary camels. In the current study, we aimed to explore the effects of vitrification of camel oocytes on mitochondrial activity, redox stress, and expression of genes related to mitochondrial function, apoptosis, pluripotency, and cytoskeleton. Moreover, we investigated developmental competence of vitrified oocytes after parthenogenetic activation. Oocytes vitrified with the Cryotop method were compared with the fresh oocytes. Our results showed that vitrification led to increased ROS production in oocytes as evidenced by an increase in the DCFDHA fluorescence intensity, and lower mitochondrial activity. At the molecular level, vitrification reduced mRNA expression of many genes, including those related to mitochondrial function (TFAM, MT-CO1, MFN1, ATP1A1, NRF1), pluripotency (SOX2 and POU5F1), and apoptosis (p53 and BAX). In contrast, expression of KLF4 and cytoskeleton-related genes (ACTB and KRT8) was not affected. However, we found no difference in the rates of oocyte survival, cleavage, and blastocyst development, and blastocyst hatching between fresh and vitrified oocytes after warming. Our results indicate that although vitrification of camel metaphase II (MII) oocytes adversely affected mitochondrial functions, the effect was transient without compromising the developmental potential of the oocytes after parthenogenetic activation.

Effect of roscovitine pretreatment on in vitro maturation of oocytes and their subsequent developmental after chemical activation in dromedary camel (Camelus dromedarius)

Studies were conducted to evaluate an optimal concentration of roscovitine needed to maintain abattoir origin oocytes at germinal vesicle stage in experiment 1 and their subsequent maturation and developmental competence after chemical activation in experiments 2 and 3, respectively. The cumulus-oocyte complexes (COCs) aspirated from ovaries collected from a local slaughterhouse were cultured in TCM-199 based pre-maturation medium supplemented with 25, 50 or 75 μM roscovitine, depending on the experimental group. After 24 h, the COCs were denuded of cumulus, fixed and stained with aceto-orcein and examined for their nuclear status. They were classified as germinal vesicle, diakinesis, metaphase-I, metaphase-II and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. In experiment 2, the COCs pre-matured in media supplemented with 50 μM roscovitine for 24 h were washed and kept for in vitro maturation along with another group of freshly collected COCs for 30 h. All the oocytes were fixed and stained to evaluate their nuclear status as described above. In experiment 3, all mature oocytes obtained from the COCs pre-matured in media supplemented with 50 μM roscovitine and those obtained from freshly collected group were activated by 5 mM ionomycin. Activated oocytes were cultured in embryo culture medium for a period of 7 days to evaluate their developmental potential. The proportion of oocytes at GV stage in the group pre-matured in media with 50 μM-was significantly (P < 0.01) higher when compared with the group having 25 μM of roscovitine. No difference was found in the proportion of GV stage oocytes in this group when compared with the freshly collected COCs. None of the oocytes reached to M-II stage in any of the three treatment groups. In experiment 2, no difference was observed in the proportion of oocytes reaching M-II stage between the groups after 30 h of in vitro culture; however, higher proportion of oocytes (P < 0.05) were classified as others in the pre-maturation group when compared with the group having freshly collected oocytes. In experiment 3, no difference was observed in the proportion of oocytes cleaving and those developing to the blastocyst stage between the pre-matured and freshly matured groups. In conclusion, the present study, for the first time, demonstrates the possible use of roscovitine as a meiotic inhibitor for camel oocytes. Keeping in view the ability of these oocytes to mature and develop to the blastocyst stage at par with the fresh oocytes, more flexible schedules for maturation and manipulation of such oocytes could be developed.

Intrafollicular spontaneous parthenogenetic development of dromedary camel oocytes.

Dromedary camel oocytes are unique in their capability forintrafollicular and in vitro spontaneous parthenogenetic activation (SPA) and development. This study was designed for (a) observing the incidence of SPA and development of dromedary camel oocytes retrieved from ovaries; (b) assessing intrafollicular development of dromedary camel oocytes using histological examination; (c) evaluating the abilities of dromedary camel oocytes to mature, SPA, and developin vitro; and (d) identifying the transcript abundance of Cdx2 messenger RNA (mRNA) expression in different stages of SPA and developed camel embryos. The results revealed that 2.33% of oocytes retrieved from dromedary camel ovaries were SPA and developed to blastocyst stage. Serial sections of dromedary camel ovaries also demonstrated the presence of 1.4 SPA and parthenotes per ovary, which included from two-cell to the blastocysts with demarcated trophectoderm and inner cell mass layers. A total of 2.6% in vitro matured dromedary camel oocytes developed into morulae.The SPA and developed dromedary embryos expressed transcript abundance for Cdx2 mRNA with the highest (p < .05) at the blastocyst. The present work determines for the first time the intrafollicular oocytes from the dromedary camel display SPA, and the parthenotes can develop into blastocysts andexpressing Cdx2 mRNA.

Effects of Short-Term Inhibition of Rho Kinase on Dromedary Camel Oocyte In Vitro Maturation.

Simple SummaryOur results revealed, for the first time, that short-term inhibition of Rho-associated protein kinases (ROCK) for 4 h prior to in vitro maturation (IVM) in a biphasic IVM approach improved oocyte nuclear maturation, producing more MII oocyte, through modulating the expression of cytokinesis- and antiapoptosis-related mRNA transcripts. This positive result suggests ROCK inhibitor as a potential candidate molecule to exploit in the control of oocyte meiotic maturation.This is the first report on a biphasic in vitro maturation (IVM) approach with a meiotic inhibitor to improve dromedary camel IVM. Spontaneous meiotic resumption poses a major setback for in vitro matured oocytes. The overall objective of this study was to improve in vitro maturation of dromedary camel oocytes using ROCK inhibitor (Y-27632) in a biphasic IVM to prevent spontaneous meiotic resumption. In the first experiment, we cultured immature cumulus–oocyte complexes (COCs, n = 375) in a prematuration medium supplemented with ROCK inhibitor (RI) for 2 h, 4 h, 6 h, and 24 h before submission to normal in vitro maturation to complete 28 h. The control was cultured for 28 h in the absence of RI. In the first phase of experiment two, we cultured COCs (n = 480) in the presence or absence (control) of RI for 2 h, 4 h, 6 h, and 24 h, and conducted real-time relative quantitative PCR (qPCR) on selected mRNA transcripts. The same was done in the second phase, but qPCR was done after completion of normal IVM. Assessment of nuclear maturation showed that pre-IVM for 4 h yielded an increase in MII oocyte (54.67% vs. 26.6% of control; p < 0.05). As expected, the same group showed the highest degree (2) of cumulus expansion. In experiment 2, qPCR results showed significantly higher expression of ACTB and BCL2 in the RI group treated for 4 h when compared with the other groups. However, their relative quantification after biphasic IVM did not reveal any significant difference, except for the positive response of BCL2 and BAX/BCL2 ratio after 4 and 6 h biphasic IVM. In conclusion, RI prevents premature oocyte maturation and gave a significantly positive outcome during the 4 h treatment. This finding is a paradigm for future investigation on dromedary camel biphasic IVM and for improving the outcome of IVM in this species.

Effect of macromolecule supplement on nuclear and cytoplasmic maturation, cryosurvival and in vitro embryo development of dromedary camel oocytes

The current evaluation of oocyte vitro maturation (IVM) media has progressed toward more defined conditions in human and livestock. In this study, the replacement of fetal calf serum (FCS) with bovine serum albumin (BSA) and polyvinyl alcohol (PVA) was evaluated during IVM in dromedary camel. Nuclear maturation rates in presence of FCS and PVA were comparable (81.6 ± 1 and 75.5 ± 5%, respectively). BSA, whether used alone or in combination with FCS, significantly reduced nuclear maturation (51.6 ± 3.9 and 54.6 ± 1.1%, respectively), compared to FCS and PVA. BSA also increased the rates of chromosome aberrations compared to FCS and PVA (25.7 ± 7.4, 8.8 ± 2.3 and 6.0 ± 2.0%, respectively). IVM macromolecule differentially affected morphological aspects of cumulus expansion and FCS promoted the highest dissociation of cumulus cells, compared to all the other groups. FCS significantly increased mean lipid intensity of oocytes compared to BSA, FCS-BSA and PVA which could explain the lower cryo-survival of oocytes matured in presence of FCS compared to BSA and PVA (56.1 ± 5.2, 91.0 ± 19.5, and 87.8 ± 6.7%, respectively). Mitochondrial activity was not affected by macromolecules, but oocytes cultured with PVA had the best redox status, compared to other IVM groups. Cleavage was not affected by IVM macromolecule, but FCS promoted significantly higher rate of morula development (51.6 ± 5.2 vs. 33.6 ± 2.9% for PVA) and blastocyst development (36.8 ± 1.4 vs. 20.5 ± 2.0% for BSA). Although adding FCS during IVM supported highest hatching rate of the resulting blastocysts, differential cell number showed no long lasting effect of IVM macromolecules on blastocyst quality. Obtained results suggest the possibility to switch from undefined to more defined IVM systems for efficient in vitro maturation and subsequent vitrification of dromedary camel oocytes. Keywords: camel, oocyte maturation, protein supplement, cryosurvival.

In vitro MATURATION OF DROMEDARY SHE-CAMEL OOCYTES EXPOSED TO LASER IRRADIATION

The objective of this study was to study the effect of laser irradiation on maturation rate of dromedary she-camel oocytes. Although in vitro fertilization (IVF) technique in she-camel has been established, but maturation rate of camel oocytes is still low comparing with other animal species. Several studies performed to improve in-vitro maturation rate using different types of media with different incubation times. In order to establish high sensitive and low cost maturation improvement technique, laser irradiation has been suggested in the present work. Cumulus oocytes complexes (COCʼs) were collected from ovaries by aspiration method and grade (A) oocytes were chosen and divided into five different groups, 62 oocytes served as control group, an un-irradiated (group 1), 64 oocytes exposed to 2 minutes of laser irradiation (group 2), 57 oocytes exposed to 3 minutes of laser irradiation (group 3), 49 oocytes exposed to 4 minutes of laser irradiation (group 4) and 52 oocytes exposed to 5 minutes of laser irradiation (group 5) with a total output power of 3 mW for different exposure durations; 2, 3, 4 and 5 minutes. Afterwards, oocytes were matured in TCM-199 medium at 38.5oC and 5% CO2 in humidified air for 42 h. Maturation rate was calculated based on expulsion of the first polar body and statistically analyzed by one way ANOVA test. The obtained results showed that, the oocytes reached germinal vesicles (GV) which exposed to laser beam for 5 minutes at 488 nm wavelength represent significantly (P<0.05) the highest value (42.31%) compared to control (not irradiated, 16.13%). However, other groups of GV showed insignificant differences with the control group. The metaphase II (M II) in the control oocytes represents significantly (P<0.05) the highest value (75.81%) compared to 3-5 minutes exposed groups. The degenerated oocytes exposed to laser beam for 5 minutes at 488 nm wavelength represent significantly (P<0.05) the highest value (40.38%) compared to control (not irradiated, 8.06%). In conclusion‚ these results indicated that the exposure of laser irradiation for 2 minutes may improve in-vitro nuclear maturation of immature oocytes in dromedary she-camels as compared to other durations (3-5 minutes) at 488 nm wavelength (blue laser).

Effects of all-trans retinoic acid on the in vitro maturation of camel (Camelus dromedarius) cumulus-oocyte complexes.

All-trans retinoic acid (RA) is a metabolite of vitamin A and has pleiotropic actions on many different biological processes, including cell growth and differentiation, and is involved in different aspects of fertility and developmental biology. In the current study, we investigated the effects of RA on camel (Camelus dromedarius) cumulus-oocyte complex in vitro maturation (IVM). IVM medium was supplemented with 0, 10, 20, and 40 µM RA. Application of 20 µM RA significantly reduced the proportion of degenerated oocytes and significantly improved oocyte meiosis and first polar body extrusion compared to the control and other experimental groups. Retinoic acid significantly reduced the mRNA transcript levels of apoptosis-related genes, including BAX and P53, and reduced the BAX/BCL2 ratio. In addition, RA significantly reduced the expression of the Transforming growth factor beta (TGFβ) pathway-related transcripts associated with the actin cytoskeleton, ACTA2 and TAGLN; however, RA increased TGFβ expression in cumulus cells. The small molecule SB-431542 inhibits the TGFβ pathway by inhibiting the activity of activin receptor-like kinases (ALK-4, ALK-5, and ALK-7); however, combined supplementation with RA during IVM compensated for the inhibitory effect of SB-431542 on cumulus expansion, oocyte meiosis I, and first polar body extrusion in activated oocytes. The current study shows the beneficial effects of RA on camel oocyte IVM and provides a model to study the multifunctional mechanisms involved in cumulus expansion and oocyte meiosis, particularly those involved in the TGFβ pathway.

Effects of cryoprotectants and cryoprotectant combinations on viability and maturation rates of Camelus dromedarius oocytes vitrified at germinal vesicle stage.

Camel fertility faces many problems, which could be solved by assisted reproductive technologies (ARTs). We designed the experiment to explore the effect of different cryoprotectant concentrations and combinations on viability and maturation rates of vitrified/warmed camel oocytes. We collected ovaries directly after slaughtering from local abattoir and transported them to laboratory in a thermo-flask containing normal physiological saline. We aspirated the oocytes from follicles, which is 2-8mm in diameter, washed three times in TCM-199 and then examined under stereo-microscope for selection. We selected morphologically normal oocytes with an evenly granulated cytoplasm and a compact cumulus cell layer. We equilibrated morphologically normal oocytes in equilibration solution (ES), which is half concentration of vitrification one. After equilibration, We transported oocytes to vitrification solution using ethylene glycol (EG, 40%), dimethyl sulphoxide (DMSO, 40%) and EG 40%+DMSO 40%. The obtained results revealed that addition of EG 40%+DMSO 40% resulted in the best quality of vitrified/warmed oocytes, which is demonstrated by higher per cent survival rate (90.16%) and maturation rate (58.95%). While DMSO 40% resulted in 62.79% and 29.54%, respectively, EG 40% reported 86.11% and 53.47%, respectively. We could conclude that vitrification of immature camel oocytes by using 40% EG+40% DMSO is suitable methods to limit drawbacks of vitrification methods, and we need further studies to assess the ability of in vitro-produced blastocyst to develop in vivo and establish pregnancy after embryo transfer.

Differences between the tolerance of camel oocytes and cumulus cells to acute and chronic hyperthermia

The dromedary camel (Camel dromedarius) is physiologically well adapted to life in hot, dry and barren land. In the present study, we report the tolerance of camel oocytes and cumulus cells to acute and chronic heat shock. Camel oocytes and cumulus cells were exposed to acute (45 °C for 2 h) and chronic (45 °C for 20 h) heat shock. Our results demonstrated that acute and chronic heat shock altered malondialdehyde concentration, which is a marker for oxidative stress. Furthermore, the heat shock reduced glutathione levels during in vitro oocyte maturation. The expression of two well-known heat shock proteins HSP70 and HSP90 were increased similarly in oocytes and cumulus cells after acute heat shock. Oocytes were less tolerant to the short acute heat shock, and showed decreased maturation, which leads to reduction in ooplasmic diameter and an increase in chromosomal count abnormalities. Furthermore, the pro-apoptotic genes P53 and BAX had increased expression levels, whereas for the anti-apoptotic gene such as BCL2 expression levels was decreased. On the other hand, the cumulus cells tolerated acute and chronic heat shock, as evident by the increase in HSP70 and HSP90 expression and steady expression levels of P53, BAX, and BCL2 after acute hyperthermia. Cumulus cells regained their vitality and ability to proliferate after chronic hyperthermia and showed wound healing capabilities after 9 days of chronic hyperthermia. Collectively, these results indicate the adaptive tolerance of camel somatic cells to acute and chronic heat shock, which is lethal to cells in many other mammals.